Anti-NME antibodies and methods for treating cancer or cancer metastases

Abstract

This invention provides anti-NME antibodies and their use in the treatment or prevention of disease. [Solution] Provide an NME7-specific antibody or fragment thereof that binds to the NME7 B3 peptide of a specific sequence.

Description

[Background technology]

[0001] 1. Field of Invention This invention relates to NME proteins, peptides derived from NME proteins, antibodies produced from these peptides, or antibodies or antibody fragments selected based on their binding ability to the peptides. The invention also relates to the treatment or prevention of diseases associated with NME expression in patients.

[0002] 2. Background Technology and Current Technology NDPK (nucleoside diphosphate protein kinase) proteins are a group of proteins that all contain an NDPK domain. The first discovered NME proteins, formerly called NM23 proteins, were NM23-H1 and NM23-H2. For decades, it was unclear whether they induce or inhibit hematopoietic cell differentiation. We previously found that NM23-H1, when in dimer form, is MUC1 * It binds to growth factor receptors and inhibits differentiation, but at higher concentrations of NM23-H1 it becomes a hexamer, which is MUC1 * It was found that it does not bind to MUC1 but induces differentiation. NM23 was called a metastasis suppressor when it was found to be underexpressed in certain highly invasive cancers. The inventors previously found that the NM23-H1 dimer is overexpressed in MUC1, which is overexpressed in most cancers. * We revealed that NM23 binds to the extracellular domain of the growth factor receptor, causing it to dimerize, and that this binding promotes the proliferation of cancer cells. Conversely, at higher concentrations, NM23 binds to MUC1 * They form tetramers and hexamers that do not bind to or promote tumorigenesis. To date, their functions have not been elucidated, but very recently, more NME family proteins (NME1-10) have been discovered. NME7 is a newly discovered NME family protein, but unlike other NME family members, its NDPK domain does not have enzymatic activity. NME7 is either not expressed at all or expressed at very low levels in adult tissues. Summary of the Invention

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

[0004] The peptide may be highly homologous to the peptides listed in FIGS. 6 to 9 (SEQ ID NOs: 88 to 145), or have 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 relative to said peptides. 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 may be polyclonal, monoclonal, bivalent, monovalent, bispecific, an antibody fragment comprising a variable domain, or an antibody mimetic. The antibody may be a human antibody or a humanized antibody. The antibody may be a single-chain scFv.

[0005] In another aspect, the present invention provides NME7 ABThe present invention relates to a method for treating or preventing a target cancer, comprising administering to the target a peptide that is highly homologous or identical to a region in the target area. The peptide may be at least 80% homologous to one or more of the peptides listed in Figure 6. The peptide may be at least 80% homologous to one or more of the peptides listed in Figure 7. The peptide may be at least 80% homologous to one or more of the peptides listed in Figure 8. The peptide may be at least 80% homologous to one or more of the peptides listed in Figure 9. The peptide may be selected from the peptides listed in Figures 6-9 (SEQ ID NOs. 88-145). The peptide may be selected from those listed in Figure 9 (SEQ ID NOs. 141-145). Alternatively, the peptides may be highly homologous to the peptides listed in Figures 6-9 (SEQ ID NOs. 88-145), or may have up to 7, 6, 5, 4, 3, 2, or 1 amino acid residues added or removed from their N-terminus or C-terminus. The peptides may be linked to other peptides via spacers or linkers.

[0006] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) for the treatment or prevention of cancer, wherein the extracellular target portion of the CAR comprises a peptide fragment of at least a member of the NME family. The NME family may be the NME7 family. A member of the NME7 family may be NME7. Or, a member of the NME7 family may be NME7 AB Or NME7 ABIt may be a similar protein. Members of the NME7 family may also be NME7-X1. The extracellular target portion of the CAR may contain one peptide from several peptides listed in Figures 6-9 (SEQ ID NOs. 88-145). The peptide may be selected from those listed in Figure 9 (SEQ ID NOs. 141-145). The peptide may be highly homologous to the peptides listed in Figures 6-9 (SEQ ID NOs. 88-145), or may have up to 7, 6, 5, 4, 3, 2, or 1 amino acid residues added or removed at the N-terminus or C-terminus. The peptide may be linked to another peptide via a spacer or linker.

[0007] In yet another aspect, the present invention relates to a method for treating or preventing cancer or cancer metastasis, comprising manipulating the chimeric antigen receptor described in claim 3 into immune system cells and administering the cells to a target requiring such manipulation.

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

[0009] In yet another aspect, the present invention relates to a method for vaccinating humans against cancer or metastatic cancer, comprising immunizing humans with peptide fragments of members 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 are NME7 AB Or NME7 ABIt may be a similar protein. The NME7 family may be NME7-X1. The immunization peptide may be one peptide from a group of peptides listed in Figures 6-9 (SEQ ID NOs. 88-145). Preferably, the peptide may be selected from those listed in Figure 9 (SEQ ID NOs. 141-145). The immunization peptide may include peptides that are highly homologous to the peptides listed in Figures 6-9 (SEQ ID NOs. 88-145), or peptides to which up to 7, 6, 5, 4, 3, 2, or 1 amino acid residues have been added or deleted at the N-terminus or C-terminus. The immunization peptide may be linked to another peptide by a spacer or linker.

[0010] In another embodiment, the present invention relates to NME7, NME7b, and NME7 AB The present invention relates to a method for treating or preventing cancer in a subject, including administering nucleic acids that suppress the expression of NME7-like proteins or NME7-X1. The nucleic acids are NME7, NME7 AB It may be an antisense nucleic acid that suppresses the expression of NME7-X1 or similar proteins. The nucleic acid is NME7, NME7 AB These may be repressive RNA, siRNA, RNAi, or shRNA that suppress the expression of a similar protein or NME7-X1.

[0011] In another embodiment, the present invention relates to NME7, NME7b, and NME7 AB The present invention relates to a method for treating or preventing cancer in a subject, comprising administering a nucleic acid that has been gene-edited to suppress the expression of a protein similar to NME7 or NME7b, NME7 AB Genetically edited nucleic acids that suppress the expression of NME7-X1, or similar proteins, can be inserted into cells that can then be administered to the patient. (NME7, NME7b, NME7) AB Genetically edited nucleic acids that suppress the expression of proteins like NME7-X1 can be inserted into cells using viral vectors. These viral vectors may be lentiviral systems.

[0012] In another embodiment, the present invention relates to NME7 AB NME7b, NME7 AB The present invention relates to a method for growing cancer cells, which involves contacting the cells with a protein similar to NME7-X1, 2i, or 5i. AB NME7b, NME7 AB Culturing cells in a medium containing NME7-like proteins or NME7-X1, 2i, or 5i, or human NME7 AB NME7b, NME7 AB A protein that expresses NME7-X1, or NME7 AB NME7b, NME7 AB The procedure involves administering a protein similar to NME7-X1 or NME7-X1 to grow cells in animals. Cancer cells may be cancer cells of the breast, prostate, ovaries, colorectal, pancreas, liver, melanoma, or brain. Drug candidates may be tested in cells. The efficacy of a drug may be evaluated by comparing cancer growth to a control without the drug, or by comparing the expression levels of metastasis markers or stem cell markers to a control without the drug, or by comparing the ability of the obtained cells to form tumors in animals from a low cell copy number to a control without the drug, and by determining the efficacy of the candidate drug for the treatment of cancer or metastasis. Cells are obtained from patients being evaluated for the treatment of cancer, and drugs that appear to be effective in those patients are selected based on the results using the methods described above. Cells are not obtained from patients being evaluated for the treatment of cancer, but drugs that appear to be effective in those patients are selected based on the results using the methods described above.

[0013] In another aspect, the present invention relates to a method for generating antibodies or antibody-like molecules from peptides or peptide mimics having sequences derived from sequences of NME. NME may be NME7. Peptides can be used as immunogens to generate antibodies or antibody-like molecules. Peptides can be administered to animals to generate anti-NME7 antibodies. Peptides can be administered to humans to generate anti-NME7 antibodies. Peptides may have sequences listed in Figures 6-9 (SEQ ID NOs. 88-145). Preferably, peptides can be selected from those listed in Figure 9 (SEQ ID NOs. 141-145). Peptides may include peptides that are highly homologous to the peptides listed in Figures 6-9 (SEQ ID NOs. 88-145), or peptides to which up to 7, 6, 5, 4, 3, 2, or 1 amino acid residues have been 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, and includes the following steps: 1) A step of obtaining a sample from a patient who has cancer or is at risk of developing cancer; 2) The step of providing 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) A step of comparing the level of a measured member of the NME7 family, or a nucleic acid encoding a member of the NME7 family, in the test sample with the level in a control patient or control cells; 4) A step of determining that the level of a member of the NME7 family, or a nucleic acid encoding a member of the NME7 family, is elevated compared to a control; and 5) A step in concluding that cancer is progressing if the donor of the test sample has cancer, or if the control on which the test sample is compared is from a donor previously diagnosed with cancer. In this method, the detection of members of the NME7 family in the circulatory system or tissue may be an indicator of cancer in the patient. Members of the NME7 family include NME7, NME7b, NME7-X1, or NME7 AB It could be a similar protein.

[0015] In yet another aspect, the present invention relates to a patient who is a member of the NME7 family or MUC1 * Steps to detect the presence of a member of the NME7 family or MUC1 * Patients exhibiting this symptom should be given anti-NME7 or anti-MUC1 * The present invention relates to a method including the step of administering an antibody or multiple antibodies. Members of the NME7 family include NME7, NME7b, NME7-X1, or NME7 AB It could be a similar protein.

[0016] In yet another aspect, the present invention relates to a method for treating or preventing cancer, including: 1) A step of obtaining a sample from a patient who has cancer, is at risk of developing cancer, or is at risk of developing metastatic cancer; 2) A step of 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) A step of determining whether the patient has cancer or has developed a more aggressive or metastatic cancer; 4) The step of administering an effective dose of a therapeutic agent to the patient that suppresses the expression of a member of the NME7 family, inhibits the cleavage of NME7, or inhibits the binding of NME7 to its target. The target of the NME7 family members is MUC1 * It is possible. The target of members of the NME7 family is MUC1 *It may be the PSMGFR portion of the extracellular domain. Members of the NME7 family are NME7, NME7b, NME7-X1, or NME7 AB It could be a similar protein.

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

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

[0019] The present invention also relates to isolated nucleic acids encoding antibodies.

[0020] The present invention also relates to isolated hybridomas expressing the monoclonal antibody described above. The antibody is NME7 AB Alternatively, it may bind specifically to NME7-X1 but not specifically to NME1. The antibody is NME7 AB and MUC1 * Interactions between extracellular domains, or between NME7-X1 and MUC1 * The interaction between extracellular domains can be disrupted. Alternatively, the antibody can disrupt NME7 AB The antibody can cleave the binding between NME7 and PSMGFR, or the binding between NME7-X1 and PSMGFR. Furthermore, the antibody can cleave NME7 AB The coupling between N-10 and N-10, or the coupling between NME7-X1 and N-10, may be broken.

[0021] In another embodiment, the antibody is NME7 AB and MUC1 * Interactions between extracellular domains, or between NME7-X1 and MUC1 * In some cases, the interaction between extracellular domains may not be disrupted. (NME7) ABAlternatively, NME7-X1 can bind to the N-10 peptide (SEQ ID NO: 170) but not to the C-10 peptide (SEQ ID NO: 171). In particular, the antibody may be 5A1, 4A3, 5D4, or 4P3.

[0022] The antibody contains the following amino acid sequence in the heavy chain variable region: YTFTNYGMN (sequence number 439) in the CDR1 region; WINTYTGEPTYVDDFKG (sequence number 440) in the CDR2 area and LRGIRPGPLAY(sequence number 441); and in the CDR3 area The amino acid sequence in the light chain variable region, including the following: SASSSVSYMN (sequence number 444) in the CDR1 region; GISNLAS(sequence number 445); and in the CDR2 region QQRSSYPPT (sequence number 446) in the CDR3 region It may include.

[0023] An example of such an antibody is 5A1.

[0024] In another embodiment, the antibody has an amino acid sequence in the heavy chain variable region including the following: NTFTEYTMH (sequence number 429) in the CDR1 region; GNFPNNGVTNYNQKFKG (sequence number 430) in the CDR2 region and RYYHSTYVFYFDS (sequence number 431) in the CDR3 region, and The amino acid sequence in the light chain variable region, including the following: SASQGISNYLN (sequence number 434) in the CDR1 region; YTSSLHS(sequence number 435) in the CDR2 area and QQYSKLPYT (sequence number 436) in the CDR3 region It may include.

[0025] An example of such an antibody is 5D4.

[0026] In another embodiment, the antibody has an amino acid sequence in the heavy chain variable region including the following: NTFTEYTMH (sequence number 388) in the CDR1 region; GNFPNNGVTNYNQKFKG (sequence number 389) in the CDR2 region and RYYHSLYVFYFDY(sequence number 390); and in the CDR3 area The amino acid sequence in the light chain variable region, including the following: SASQGISNYLN (sequence number 393) in the CDR1 region; YTSSLHS(sequence number 394); and in the CDR2 area QQYSKLPYT (sequence number 395) in the CDR3 region It may include.

[0027] An example of such an antibody is 4P3.

[0028] In another embodiment, the antibody has an amino acid sequence in the heavy chain variable region including the following: NTFTEYTMH (sequence number 388) in the CDR1 region; GNFPNNGVTNYNQKFKG (sequence number 389) in the CDR2 region and RYYHSLYVFYFDY(sequence number 390); and in the CDR3 area The amino acid sequence in the light chain variable region, including the following: ITSTDIDDDMN(sequence ID) in the CDR1 area; EGNTLRP(sequence number); and in the CDR2 region LQSDNLPLT(sequence ID) in the CDR3 region It may include.

[0029] An example of such an antibody is 4P3.

[0030] The antibody may be a human antibody, a humanized antibody, or a modified antibody mimetic.

[0031] The antibodies may be non-human antibodies, such as those from mice or camels.

[0032] 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 containing the above-mentioned antibody.

[0033] 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 containing the above-mentioned antibody.

[0034] 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 antibody and comparing the results obtained from both samples, wherein the presence of positive binding to the antibody in the patient sample indicates the presence of cancer or cancer metastasis in the patient. The antibody may be bound to an imaging agent. The patient sample may be blood, body fluid, tissue, or circulating cells, in vitro or in vivo, including intraoperatively.

[0035] This invention also addresses anti-NME7 AB This relates to cells that are manipulated to express antibodies or fragments 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.

[0036] Cells may contain chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Expression of anti-NME7 antibodies may be inducible. AB The nucleic acid encoding the antibody can be inserted into the Foxp3 enhancer or promoter. (Anti-NME7) AB The antibody may be present in the NFAT induction system. The NFATc1 response element may be inserted upstream of the antibody sequence, which is inserted into the enhancer or promoter region.

[0037] anti-NME7 AB The antibody or its fragment may be able to bind to the NME7 B3 peptide, or NME7 AB Or, MUC1 of NME7-X1 *This can disrupt the binding of the extracellular domain to PSMGFR.

[0038] CAR can recognize tumor-associated antigens and anti-NME7 antibodies. Tumor-associated antigens include MUC1. * It is possible.

[0039] The present invention also includes NME7 listed in Figures 6 to 9 as the immunogenicity-inducing moiety. AB The present invention relates to an anti-cancer vaccine comprising a composition containing one or more peptides derived from the same, or a peptide having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto. The peptide may be the peptides of SEQ ID NOs. 141 to 145, or a peptide having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto. The peptide may be SEQ ID NO. 145, or a peptide having 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0040] In another embodiment, the present invention relates to a BiTE comprising the above-mentioned antibody.

[0041] In yet another aspect, the present invention relates to anti-NME7 AB Regarding the method for generating antibodies, the cysteine ​​residue of the NME7 B3 peptide is mutated to avoid disulfide bonds.

[0042] In yet another embodiment, the present invention relates cells to NME7 AB The present invention relates to a method for generating cells with enhanced metastatic potential, including culturing them with NME7-X1.

[0043] This invention also relates to NME7 AB Alternatively, cells engineered to express NME7-X1, NME7 AB Or, with respect to a genetically modified animal expressing NME7-X1, NME7 AB Alternatively, NME7-X1 could be human, and NME7 AB Alternatively, NME7-X1 expression may be inducible.

[0044] This patent or application file includes at least one color drawing. A copy of this patent or patent application publication, including the color drawing, will be provided by the Patent Office upon request, upon payment of the required fees. The present invention may be better understood from the detailed description given below and the accompanying drawings provided for illustrative purposes only, but this shall not limit the invention. [Brief explanation of the drawing]

[0045] [Figure 1] This graph shows the HRP signal obtained from an ELISA sandwich assay, demonstrating that NME7AB dimerizes the MUC1* extracellular domain peptide. [Figure 2] This graph shows RT-PCR measurement of stem cell markers and cancer stem cell marker gene expression in T47D cancer cells after culturing in conventional culture medium or NME7-containing medium. Cells that became non-adherent (suspended) were separated from those that remained adhered and analyzed. [Figure 3] This is a graph of RT-PCR measurements of gene expression for various stem and putative cancer stem cell markers in DU145 prostate cancer cells. Cells were cultured in either conventional medium or medium containing either NME1 dimer ("NM23") or NME7 (NME7AB). Rho kinase inhibitors were not used because the cells remained attached after two passages. [Figure 4] RT-PCR measurements of metastasis and pluripotent stem cell markers show that 2i inhibitors (GSK3 beta and MEK inhibitors), which have been previously shown to return stem cells to a more naive state, also induce cancer cells to a more metastatic state, though not to the same extent as NME7AB. [Figure 5] This is a sequence alignment between human NME1 and human NME7-A or -B domains. [Figure 6-1] This list includes human NME7-derived immunogenic peptides selected for their low sequence identity to NME1 and their ability to generate therapeutic anti-NME7 antibodies for the treatment or prevention of cancer. [Figure 6-2] This list includes human NME7-derived immunogenic peptides selected for their low sequence identity to NME1 and their ability to generate therapeutic anti-NME7 antibodies for the treatment or prevention of cancer. [Figure 7] This list includes human NME7-derived immunogenic peptides selected for their ability to generate therapeutic anti-NME7 antibodies for the treatment or prevention of cancer, which may be important for structural integrity or binding to MUC1*. [Figure 8] This list enumerates human NME1-derived immunogenic peptides selected for their ability to generate therapeutic anti-NME7 antibodies for the treatment or prevention of cancer, which may be important for structural integrity or binding to MUC1*. [Figure 9] We list immunogenic peptides derived from human NME7, selected due to their low sequence identity to NME1 and their homology to bacterial NME1 proteins that have been associated with cancer. These peptides are more preferred due to their ability to produce therapeutic anti-NME7 antibodies for the treatment or prevention of cancer. The peptides shown in this figure contain an additional cysteine ​​covalently bonded at the C-terminus. [Figure 10] The graphs show ELISA assays in which NME7-AB (Figure 10A) or NME1 (Figure 10B) were adsorbed onto a plate, and anti-NME7 antibodies generated by NME7 peptides A1, A2, B1, B2, and B3 were tested for their ability to bind to NME7 but not to NME1. C20 is the anti-NME1 antibody. [Figure 11] The graph shows the results of an ELISA assay that tested the generated anti-NME7 antibodies regarding their ability to inhibit the binding of NME7-AB to the surface-immobilized MUC1* peptide, but not to the binding of NME1. [Figure 12]The graphs show cancer cell proliferation experiments in which breast cancer cells were grown in the presence or absence of NME7 antibodies or short-chain peptides derived from NME7, which were used to generate or select antibodies. Furthermore, antibodies generated by immunization with amino acids 100-376, which is almost the entire NME7AB peptide, were shown to suppress cancer cell proliferation. [Figure 13] The graph shows cancer cell proliferation experiments in which breast cancer cells were grown in the presence or absence of combinations of NME7 antibodies or short-chain peptides derived from NME7, which were used to generate or select antibodies. Both antibodies, as well as their immunized NME7AB peptides, suppressed the proliferation of cancer cells. [Figure 14] This table shows scientists' observations of cancer cells growing in NME7-AB or 2i inhibitors, both of which can transform cancer cells into a more metastatic state, and in the presence or absence of NME7-derived peptides A1, A2, Bl, B2, and B3. The NME7-AB peptide suppressed the migration of adherent cancer cells to suspension cells, and RT-PCR measurements showed increased expression of metastasis markers, particularly CXCR4. [Figure 15A] Graphs of RT-PCR measurements of CXCR4 and other metastasis markers in T47D breast cancer cells grown in NME7-AB or 2i inhibitors, respectively, which transform cancer cells into a more metastatic state, and the inhibitory effect of anti-NME7 antibodies on metastatic transformation are shown. Figure 15A is a PCR graph of CXCR4 expression in T47D cancer cells grown in NME7AB or 2i in the presence or absence of anti-NME7 antibody. [Figure 15B]Graphs of RT-PCR measurements of CXCR4 and other metastasis markers in T47D breast cancer cells grown in either NME7-AB or 2i inhibitors, respectively, which transform cancer cells into a more metastatic state, and the inhibitory effect of anti-NME7 antibodies on metastatic transformation are shown. Figure 15B is a graph of RT-PCR measurements of CXCR4, CHD1, and SOX2 expression in T47D breast cancer cells grown for 72 or 144 hours in the presence of NME7AB immunized peptides and 2i inhibitors, showing that these peptides themselves are inhibitory against metastatic transformation. Peptides A1, A2, and B1 used in inhibitory combos 2 and 3 in Figure 15A are also inhibitory as peptides. Peptide B3 is the most inhibitory and is the immunized peptide for antibody 61, which was the most inhibitory antibody tested in Figure 15A. [Figure 15C] The graphs show RT-PCR measurements of CXCR4 and other metastasis markers in T47D breast cancer cells grown in NME7-AB or 2i inhibitors, respectively, which transform cancer cells into a more metastatic state, and the inhibitory effect of anti-NME7 antibodies on metastatic transformation. Figure 15C is an enlarged graph of the Y-axis in Figure 15B. [Figure 16] The table shows the threshold cycle number for control housekeeping genes and CXCR4 expression, as well as the recorded RNA levels in the samples used for CXCR4 RT-PCR measurement in Figure 31. [Figure 17] The graphs show the RT-PCR measurements of NME7-X1 expression in a series of human stem cells and cancer cells. [Figure 18] This graph shows RT-PCR measurements of NME7, NME7a, NME7b, and NME7-X1 expression in a series of human stem cells and cancer cells. NME7a is full-length NME7, NME7b has a small deletion in the DM10 domain, and NME7-X1 has all of the DM10 domain and a small portion of the N-terminus of the first NDPK A domain. Bars labeled NME7 indicate that primers were used to detect both NME7a and NME7b. [Figure 19-1]Figure 19A shows Western blot images in which various cancer cell lines are detected for NME7 expression using antibodies produced by immunization with NME7-derived peptides. Figure 19A shows a Western blot using antibody 52 bound to peptide A1 to detect the presence of full-length NME7, NME7AB, or NME7-X1 in a series of cells. Figure 19B shows a Western blot using antibody 56 bound to peptide B1 to detect the presence of full-length NME7, NME7AB, or NME7-X1 in a series of cells. Figure 19C shows a Western blot using antibody 61 bound to peptide B3 to detect the presence of full-length NME7, NME7AB, or NME7-X1 in a series of cells. [Figure 19-2] Figure 19 shows a Western blot image in which various cancer cell lines are detected for expression of NME7 species using antibodies produced by immunization with NME7-derived peptides. Figure 19D shows a Western blot using the commercially available polyclonal antibody H278, produced against both NME7 A and B, to detect the presence of full-length NME7 in a series of cells. As the figure shows, antibody H278 also recognizes NME1. Figure 19E shows a gel published on the website for the commercially available anti-NME7 antibody B9, showing that it binds to a species with the apparent molecular weight of full-length NME7. Figure 19F shows a Western blot using anti-NME7 antibody B9 to detect a gel filled with only NME1. As seen in the figure, antibody B9 recognizes both NME1 and full-length NME7. This is not surprising because, like antibody H278, B9 is produced against both the A and B domains of NME7, and the A domain of NME1 is highly homologous to the A domain of NME7AB. [Figure 20-1]Figure 20A shows RT-PCR measurement graphs of metastasis markers in cancer cells cultured in serum-free medium containing NME7AB compared to standard medium. Figure 20A shows that the MUC1-positive ovarian cancer cell line SK-OV3 increased the expression of the metastasis markers CXCR4, CDH1 (also known as E-cadherin), SOX2, and NME7-X1. Figure 20B shows that the MUC1-negative ovarian cancer cell line OV-90 increased the expression of the metastasis markers CXCR4 and NME7-X1. [Figure 20-2] Figure 20C shows RT-PCR measurement graphs of metastasis markers in cancer cells cultured in serum-free medium containing NME7AB compared to standard medium. MDA-MB, a breast cancer cell line expressing the lowest levels of MUC1, showed increased expression of the metastasis markers CDH1, also known as E-cadherin, and SOX2. [Figure 21-1] Photographs and descriptions of the Western blots of the analyzed cancer cell lines are shown. For the Western blots in Figures 21A and 21B, all cancer samples were normalized so that they were packed into the gel at a concentration of 40 ug / mL. In Figure 21A, the expression of full-length MUC1 in various cancer cell lines is detected using the anti-tandem repeat monoclonal antibody VU4H5. In Figure 21B, the expression of truncated MUC1* is detected in various cancer cell lines using the polyclonal anti-PSMGFR antibody. Figure 21C is a description of the analyzed cancer cell lines. [Figure 21-2]Photographs and descriptions of the Western blots of the analyzed cancer cell lines are shown. Figure 21D shows that while HER2 expression levels remained unchanged, MUC1* expression dramatically increased as the cells acquired resistance to Herceptin. Figure 21E shows a graph of the proliferation of parental BT474 cells compared to drug-resistant metastatic cells in response to Herceptin treatment in the presence or absence of anti-MUC1* Fab. As seen in the figure, BT474 parental cells showed a Herceptin concentration-dependent reduction in cell proliferation, while two Herceptin-resistant cell lines, BTres1 and BTres2, did not show a reduction in cancer cell proliferation in response to Herceptin treatment. However, when treated with anti-MUC1* Fab, the resistant cell lines showed a Herceptin concentration-dependent reduction in cancer cell proliferation. Figure 21F shows a graph of percentage cell death in parental BT474 cells in response to treatment with Taxol, in or without anti-MUC1* Fab, compared to drug-resistant BTRes1 cells. [Figure 22] The images show Western blots from co-immunoprecipitation experiments. T47D breast cancer cell extracts were incubated with either the antibody against the cytoplasmic end of MUC1, Ab-5, or the control antibody, IgG, and co-immunoprecipitation was performed. The gels were blotted with two different commercially available anti-NME7 antibodies, B9 (Figure 22A) and CF7 (Figure 22B). Both gels showed characteristic NME7 bands at approximately 33 kDa and 30 kDa, respectively. The gels were stripped and re-detected with the antibody against the extracellular domain of MUC1*, anti-PSMGFR ((Figure 22C) and (Figure 22D)). This indicates that NME7 species and MUC1* interact. Recombinant NME7-AB and recombinant NME7-X1 were mixed together, spread on a gel, and then detected with anti-NME7 antibody. This study demonstrates that two unique NME7 species spontaneously arise in breast cancer cells and interact with MUC1*: NME7-AB-like and NME7-X1 (Figure 22E). [Figure 23]The images show 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 end of MUC1, Ab-5, or a control antibody, IgG, and co-immunoprecipitation was performed. The gels were blotted with commercially available anti-NME7 antibody B9 (Figure 23A). Both cell types showed characteristic NME7 bands at approximately 33 kDa and 30 kDa. The gels were stripped and re-detected with an antibody against the extracellular domain of MUC1*, anti-PSMGFR (Figure 23B). This indicates that NME7 species and MUC1* interact. Recombinant NME7-AB and recombinant NME7-X1 were mixed, spread on the gel, and then detected with anti-NME7 antibody. This study demonstrates that two unique NME7 species spontaneously arise in breast cancer cells and interact with MUC1*: NME7-AB-like and NME7-X1 (Figure 23C). [Figure 24] This graph shows the results of an ELISA experiment assaying the ability of novel anti-NME7 antibodies to bind to NME7-AB. NME7-AB is known to bind to the extracellular domain of MUC1*. The surface of a multiwell plate was coated with recombinant NME7-AB. Anti-NME7-AB antibodies were added separately to the wells. Standard washing was performed, and visualization was achieved by adding an HRP-labeled secondary antibody. As can be seen from the figure, 7 out of 10 anti-NME7 antibodies bound strongly to NME7-AB. [Figure 25] This graph shows the results of an ELISA experiment assaying the ability of novel anti-NME7 antibodies to bind to NME1, or, preferably, their inability to bind. The surface of a multi-well plate was coated with recombinant NME1-S120G dimer, known to bind to the MUC1* extracellular domain. Anti-NME7-AB antibody was added separately to the wells. Standard washing was performed, and visualization was achieved by adding an HRP-labeled secondary antibody. As can be seen from the figure, only one antibody showed minimal binding to NME1. [Figure 26]The graph shows the results of an ELISA competitive inhibition assay. After constructing the NME7-AB / anti-NME7 antibody conjugate, it was added to a multi-well plate coated with PSMGFR and the MUC1* extracellular domain peptide. It should be noted that NME7-AB has two pseudo-identical domains, A and B, that can bind to the MUC1* extracellular domain, respectively. The antibody that binds to the NME7 B3 peptide in the B domain does not bind to the NME7 A domain. Therefore, only partial inhibition of the NME7-AB / MUC1* interaction is predicted. [Figure 27] The graph shows the results of the ELISA substitution assay. NME7-AB was first bound to the surface-immobilized MUC1* extracellular domain peptide on the plate, and then fragmented by the addition of an anti-NME7 antibody. [Figure 28] The graph of the ELISA substitution assay is shown. In this case, a multiwell plate was coated with the shortened MUC1* peptide, N-10 (which has 10 N-terminal amino acid deletions in the PSMGFR sequence). NME7-AB is known to bind to the N-10 peptide. NME7-AB was bound to the surface-immobilized N-10 peptide on the plate and then fragmented by the addition of an anti-NME7 antibody. [Figure 29] The graphs show the amount of RNA present in samples of T47D breast cancer cells cultured in either the standard recommended medium, RMPI, serum-free medium containing only 4 nM (optimal value) or 8 nM of NME7-AB as a growth factor, or serum-free medium containing only 8 nM of NME1 S120G dimer as a growth factor. Since NME1 is a homodimer and NME7-AB is a monomer consisting of two pseudo-identical domains, 8 nM of NME1 is the molar equivalent of 4 nM of NME7-AB. Cancer cells were cultured in or without anti-NME7 B3 antibody. In this experiment, suspension cells were isolated from adherent cells and analyzed separately. Significant data indicate that suspension cells are cancer stem cells. Increases or decreases in the amount of RNA in the sample indicate the number of cells with increased or decreased RNA, respectively, in a given population. [Figure 30]The graphs show PCR measurements of the metastasis marker CXCR4 in T47D breast cancer cells cultured in either the standard recommended medium, RPMI, serum-free medium containing only 4nM (optimal value) or 8nM NME7-AB as a growth factor, or serum-free medium containing only 8nM NME1 S120G dimer as a growth factor. Since NME1 is a homodimer and NME7-AB is a monomer consisting of two pseudo-identical domains, 8nM NME1 is the molar equivalent of 4nM NME7-AB. Cancer cells were cultured in or without anti-NME7 B3 antibody. In this experiment, suspension cells were isolated from adherent cells and analyzed separately. Significant data indicate that suspension cells are cancer stem cells. As can be seen from the figure, proliferation in NME7-AB medium increased CXCR4 in the suspension cell population, anti-NME7 B3 antibody reduced expression, and anti-NME7 antibody reduced the generation of cancer stem cells. [Figure 31] The graphs show PCR measurements of stem cell markers and metastasis markers in T47D breast cancer cells cultured in either the standard recommended medium, RMPI, serum-free medium containing only 4nM (optimal value) or 8nM NME7AB as a growth factor, or serum-free medium containing only 8nM NME1 S120G dimer as a growth factor. Since NME1 is a homodimer and NME7-AB is a monomer consisting of two pseudo-identical domains, 8nM NME1 is the molar equivalent of 4nM NME7-AB. Cancer cells were cultured in or without anti-NME7 B3 antibody. In this experiment, suspension cells were isolated from adherent cells and analyzed separately. Significant data indicate that suspension cells are cancer stem cells. As can be seen from the figure, proliferation in NME7-AB medium increased SOX2 expression in the suspension cell population, anti-NME7 B3 antibody reduced expression, and anti-NME7 antibody reduced the generation of cancer stem cells. [Figure 32]The graphs show PCR measurements of stem cell markers and metastatic growth factor receptor MUC1 in T47D breast cancer cells cultured in either the standard recommended medium, RMPI, serum-free medium containing only 4nM (optimal value) or 8nM NME7-AB as a growth factor, or serum-free medium containing only 8nM NME1 S120G dimer as a growth factor. Since NME1 is a homodimer and NME7-AB is a monomer consisting of two pseudo-identical domains, 8nM NME1 is the molar equivalent of 4nM NME7-AB. Cancer cells were cultured in or without anti-NME7 B3 antibody. In this experiment, suspension cells were isolated from adherent cells and analyzed separately. Significant data indicate that suspension cells are cancer stem cells. As can be seen from the figure, proliferation in NME7-AB medium increased MUC1 expression in the suspended cell population, anti-NME7 B3 antibody reduced expression, and anti-NME7 antibody reduced the generation of cancer stem cells. [Figure 33] Figure 33A shows an IVIS image of a mouse injected with 500,000 T47D-wt breast cancer cells six days after tail vein injection of cancer cells into an immunocompromised nu / nu mouse. Figure 33B shows an IVIS image of a mouse injected with 10,000 T47D breast cancer cells grown for 10 days in minimal medium NME7-AB. Suspended cells were collected. These suspended cells are referred to as cancer stem cells (CSCs) in this specification. As can be seen from the figures, mice injected with wild-type cancer cells show no signs of metastasis. However, mice injected with cancer stem cells, although 50 times fewer in number, clearly show that the injected cancer cells are metastatic. [Figure 34]Figure 34A shows IVIS images of mice injected with 500,000 T47D-wt breast cancer cells 10 days after tail vein injection of cancer cells from immunocompromised nu / nu mice. Figure 34B shows IVIS images of mice injected with 10,000 T47D-CSCs (cancer stem cells). Figure 34C shows IVIS images of mice injected with 10,000 T47D-CSCs (cancer stem cells) and then injected with anti-NME7 antibody on day 7. Figure 34D shows handwritten records of IVIS measurements of emitted photons. As can be seen from the figures, the mice selected for treatment were more metastatic than comparable T47D-CSC mice. The efficacy of the first antibody injection may have been blocked by the injection of free NME7-AB on day 6. Control mice injected with 500,000 T47D-wt cells exhibit somewhat weaker photon emission, which may be background or from surviving cancer cells. [Figure 35] Figure 35A shows an IVIS image of a mouse injected with 500,000 T47D-wt mammary cancer cells 12 days after tail vein injection of cancer cells from immunocompromised nu / nu mice. Figure 35B shows a mouse injected with 10,000 T47D-CSCs (cancer stem cells), not treated with anti-NME7 antibody, and died due to excessive tumor volume before IVIS image acquisition. Figure 35C shows an IVIS image of a mouse injected with 10,000 T47D-CSCs (cancer stem cells) and injected with anti-NME7 antibody on days 7 and 10. As can be seen from the figures, the mice treated with anti-NME7 antibody have had their cancer metastases eliminated. Control mice injected with 500,000 T47D-wt cells show less release and fewer cancer cells, or may be background. [Figure 36]Figure 36A shows IVIS images of mice injected with 500,000 T47D-wt breast cancer cells 14 days after tail vein injection of cancer cells from immunocompromised nu / nu mice. Figure 36B shows IVIS images of mice injected with 10,000 T47D-CSCs (cancer stem cells) and injected with anti-NME7 antibody on days 7, 10, and 12. As can be seen from the figures, mice treated with anti-NME7 antibody are almost completely free of cancer metastasis. Control mice injected with 500,000 T47D-wt cells show no photon emission. [Figure 37] Figures 37A, 37C, 37E, 37G, 37I, 37K, 37M, and 37O show IVIS images of mice injected into the tail vein with 500,000 T47D-wt cells on day 0. Figures 37B, 37D, 37F, 37H, 37J, 37L, 37N, and 37P show IVIS images of mice injected into the tail vein with 10,000 T47D cancer stem cells on day 0, administered anti-NME7 antibody from day 7 to day 17, where treatment was interrupted, and then resumed on day 21. Figures 37Q, 37R, 37S, 37T, and 37U show enlarged IVIS images of treated mice from day 17 to day 26 when anti-NME7 antibody therapy was interrupted, and again on day 21 when antibody therapy was resumed. Figure 37V shows the scale bar of IVIS measurements. As can be seen from this time course, cancer cells proliferating in NME7 readily metastasize, and such metastases can be effectively treated, prevented, or reversed by treatment with antibodies that bind to NME7. [Figure 38] Figure 38A shows mice injected into the tail vein (iv). Figure 38B shows mice injected into the intraperitoneal cavity (ip). Figure 38C shows mice injected subcutaneously (sc). [Figure 39]This figure shows human lung tissue samples stained with an anti-NME7 antibody that binds to the B3 peptide. This figure illustrates the absence of NME7 expression in normal tissue, the increased expression of NME7 associated with tumor malignancy, and the increased metastasis. [Figure 40] This figure shows human small intestinal tissue samples stained with an anti-NME7 antibody that binds to the B3 peptide. This figure illustrates the absence of NME7 expression in normal tissue, the increased expression of NME7 associated with tumor malignancy, and the increased metastasis. [Figure 41] This figure shows human colon tissue samples stained with an anti-NME7 antibody that binds to the B3 peptide. This figure illustrates the absence of NME7 expression in normal tissue, the increased expression of NME7 associated with tumor malignancy, and the increased metastasis. [Figure 42] Figures 42A–42C show IVIS images of female nu / nu mice, each weighing approximately 20g, that were injected into the tail vein with 10,000 luciferase-positive T47D metastatic breast cancer stem cells and treated with anti-NME7AB antibody 4A3 (also known as 8F9A4A3). To image the cancer cells, the luciferase substrate luciferin was injected intraperitoneally into the IVIS detector 10 minutes prior to imaging. Figures 42A–42C show IVIS images of animals facing downwards. Figures 42D–42F show IVIS images of animals facing upwards. Figures 42A and 42D show control animals injected with phosphate-buffered saline. Figures 42B and 42E show a prophylactic model in which animals were injected with anti-NME7AB antibody 4A3 24 hours prior to injection of metastatic cancer cells, and then received a total of 12 antibody injections over 22 days, approximately every other day. Figures 42C and 42F show a recovery model in which animals were injected with anti-NME7AB antibody 4A3 24 hours after injection of metastatic cancer cells, and then received a total of 11 antibody injections over 20 days, approximately every other day. [Figure 43]Figures 43A–43C show IVIS images of female nu / nu mice weighing approximately 20g each, in which 10,000 luciferase-positive T47D metastatic breast cancer stem cells were injected into the tail vein and treated with either anti-NME7AB antibody 5A1 (also known as 8F9A5A1) or 5D4 (also known as 5F3A5D4). To image the cancer cells, the luciferase substrate luciferin was intraperitoneally injected into the IVIS detector 10 minutes prior to imaging. Figures 43A–43C show IVIS images of animals facing downwards. Figures 43D–43F show IVIS images of animals facing upwards. Figures 43A and 43D show control animals injected with phosphate-buffered saline. Figures 43B and 43E, 43C and 43F show a prophylactic model in which animals were injected with anti-NME7AB antibody 24 hours prior to injection of metastatic cancer cells, and then received a total of 12 antibody injections over 22 days, approximately every other day. The image was obtained on the 27th day. [Figure 44]The image shows female nu / nu mice injected into the tail vein on day 0 with 10,000 luciferase-positive T47D metastatic breast cancer stem cells mixed with NME7AB at a final concentration of 32 nM. On days 1 and 2, the animals were further injected into the tail vein with 32 nM of NME7AB, which showed an increase in metastasis. This is a system to demonstrate the recovery of previously occurring metastases. On day 7, the animals were treated with individual anti-NME7AB antibodies: 8F9A5A1, 8F9A4A3, or 5F3A5D4. Figure 44A shows control animals injected with phosphate-buffered saline. Figure 44B shows animals treated with the anti-NME7AB monoclonal antibody 8F9A5A1 (also known as 5A1). Figure 44C shows animals treated with the anti-NME7AB monoclonal antibody 8F9A4A3 (also known as 4A3). Figure 44D shows animals treated with the anti-NME7AB monoclonal antibody 5F3A5D4 (also known as 5D4). Green arrows indicate resistance doses (5-7 mg / kg) over the indicated period, and red arrows indicate high doses (15 mg / kg). As can be seen from the figure, animals treated with anti-NME7AB antibody have fewer metastases than control animals, even when many animals in the group to be treated with the antibody have more metastases before treatment. Higher concentrations of anti-NME7AB antibody are more effective than lower concentrations. For example, when animals were treated with a high dose between days 11 and 17, most of the treated animals cleared metastases by approximately day 17. However, one type of low-dose antibody resulted in metastatic recurrence. The animals responded again to high-dose treatment by day 32. [Figure 45]Figure 45A shows an IVIS image of a female nu / nu mouse that received a subcutaneous injection into its right flank on day 0 of 10,000 luciferase-positive T47D metastatic breast cancer stem cells, which were mixed with NME7AB at a final concentration of 32 nM and then mixed with Matrigel in a 1:1 vol:vol ratio. Tumor engraftment was allowed to progress from day 0 to day 6. The animals were then IV-treated by tail vein infusion of anti-NME7AB antibody. Control animals were infused with PBS. Figure 45A shows an IVIS image of a control animal. Figure 45B shows an IVIS image of an animal that received a tail vein infusion of a cocktail of anti-NME7AB antibodies 5A1, 4A3, and 5D4 at a total concentration of 15 mg / kg. The antibody or PBS was administered four times between days 7 and 18. As can be seen from the figures, mice treated with anti-NME7AB antibody showed fewer metastases than the control group. In the treatment group, 2 out of 5 animals had primary tumors larger than those in the control group. This is likely because the anti-NME7AB antibody inhibited the spread of cancer cells, and therefore they remained concentrated in the primary tumor. In this experiment, PCR analysis showed that after 11 days in cultures containing NME7AB, T47D breast cancer cells showed upregulation of CXCR4 by 10⁹ times, OCT4 by 2 times, NANOG by 3.5 times, and MUC1 by 2.7 times. [Figure 46]The image shows female nu / nu mice that received subcutaneous injection into their right flank on day 0 of 10,000 luciferase-positive T47D metastatic breast cancer stem cells, which were mixed with NME7AB at a final concentration of 32 nM and then mixed with Matrigel in a 1:1 vol:vol ratio. Tumor engraftment was allowed to progress from day 0 to day 6. The animals were then IV-treated by tail vein infusion of anti-NME7AB antibody. Control animals were injected with PBS. On day 38, the animals were sacrificed, their livers were harvested, and IVIS analysis was performed 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-NME7AB antibody 5A1. Figures 46E and 46F show whole-body IVIS images of control animals injected with anti-NME7AB antibody 4A3. Figures 46G and 46H show whole-body IVIS images of control animals injected with anti-NME7AB antibody 5D4. Figures 46A, 46C, 46E, and 46G are IVIS images taken 7 days before treatment. Figures 46B, 46D, 46F, and 46H are IVIS images taken 31 days after anti-NME7AB antibody treatment or mock treatment. As can be seen from the figures, animals in the PBS control group showed metastasis in whole-body IVIS images (blue dots), while animals treated with anti-NME7AB antibody did not show metastasis. Figures 46I-46P show images and IVIS images of the liver and lungs taken from animals after sacrifice. Figures 46I, 46K, 46M, and 46O are normal images. Figures 46J, 46L, 46N, and 46P are IVIS images that reveal metastatic cancer cells. As can be seen from the figures, anti-NME7AB antibody significantly suppressed metastasis to the liver. The liver is a primary site of breast cancer metastasis. Figure 46Q is a bar graph of measured photons emitted and counted by an IVIS detector in livers collected from control animals and treated animals. [Figure 47]Figure 47A shows images of immunofluorescence experiments in which various cancer cell lines are stained to detect the presence of NME7AB. Figure 47B shows ZR-75-1 breast cancer cells (also known as 1500s) stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47C shows H1975 non-small cell lung cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47D shows H292 non-small cell lung cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47E shows HPAFII pancreatic cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47F shows DU145 prostate cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. As can be seen from the figure, all cancer cell lines tested by the inventors showed strong membrane staining against NME7AB. The monoclonal antibody used in these experiments was 5D4. In parallel, the same cell lines were stained using NME7AB antibodies 5A1 and 4A3, and the same results were obtained. [Figure 48] This section shows photographs of immunofluorescence experiments in which various human lung cancer cell lines are stained to detect the presence of NME7AB. Figures 48A–48C show H1975 non-small cell lung cancer cells, an adenocarcinoma, stained with various concentrations of anti-NME7AB antibody 5D4. Figure 48A is an overlay of DAPI and anti-NME7AB staining. Figure 48B shows anti-NME7AB staining only. Figure 48C is a magnified view of the DAPI and anti-NME7AB staining overlay. Figures 48D–48F show H292 non-small cell lung cancer cells, a mucoepidermoid carcinoma, stained with various concentrations of anti-NME7AB antibody 5D4. Figure 48D is an overlay of DAPI and anti-NME7AB staining. Figure 48E shows anti-NME7AB staining only. Figure 48F is a magnified view of the DAPI and anti-NME7AB staining overlay. Figures 48G–48I show H358 non-small cell lung cancer cells, which are metastatic bronchoalveolar carcinoma, stained with various concentrations of anti-NME7AB antibody 5D4. Figure 48G is an overlay of DAPI and anti-NME7AB staining. Figure 48H shows anti-NME7AB staining only. Figure 48I is a magnified view of the DAPI and anti-NME7AB staining overlay. [Figure 49-1]The PCR graphs of cancer cell lines—breast cancer T47D, lung H1975, lung H358, and pancreatic HPAFII—before and after culture in NME7AB are shown. Figure 49A shows the measured breast cancer metastasis marker CXCR4. Figure 49B shows the measured stem cell marker OCT4. Figure 49C shows the measured metastasis marker ALDH1. Figure 49D shows the measured stem cell marker SOX2. Figure 49E shows the measured stem cell marker NANOG. Figure 49F shows the measured metastasis marker CDH1 (also known as E-cadherin). Animals injected with cancer stem cells are those injected with NME7AB-proliferating suspension cells. As can be seen from the figures, metastasis markers, stem cell markers, or markers of epithelial-mesenchymal transition (EMT) are increased after culture in NME7AB, indicating a transition to a higher metastatic state. [Figure 49-2] PCR graphs of cancer cell lines—breast cancer T47D, lung H1975, lung H358, and pancreatic HPAFII—before and after culture in NME7AB are shown. Figure 49G shows the measured metastasis marker CD133. Figure 49H shows the measured stem cell marker ZEB2. Figure 49I shows the measured stem, cancer, and metastasis marker MUC1. Suspension cells (also known as tumor spheres) become independently able to proliferate scaffolds and show increased metastasis markers compared to adherent cells. Animals injected with cancer stem cells are animals injected with NME7AB-proliferating suspension cells. As can be seen from the figures, metastasis markers, stem cell markers, or markers of epithelial-mesenchymal transition (EMT) are increased after culture in NME7AB, indicating a transition to a higher metastatic state. [Figure 50]Figures 50A and 50C show IVIS images of NSG mice injected into the tail vein with 10,000 cancer cells, either NCI-H358 parental cells or NCI-H358 cells, after 10 days in a culture containing NME7AB. Figures 50A and 50C show IVIS images of mice injected with NCI-H358 lung cancer cells grown in NME7AB for 10 days. Figures 50B and 50D show IVIS images of mice injected with parental NCI-H358 cells. Figures 50A and 50B show IVIS images of mice acquired facing downwards. Figures 50C and 50D show IVIS images of mice acquired facing upwards. As can be seen from the figures, NME7AB-grown cells showed significantly increased metastatic potential. [Figure 51] The graphs show PCR results for the MUC1-negative prostate cancer strain PC3 before and after 2 or 3 passages in culture in dimer NM23-H1 (also known as NME1) or NME7AB. The graphs show the multiplicative differences in stem cell, cancer cell markers, and metastasis markers. As can be seen from the figure, repeated culture in NME1 or NME7AB induces upregulation of stem, cancer, and metastasis markers, as well as 5-8-fold upregulation of MUC1 expression. [Figure 52]The image shows female nu / nu mice injected into the tail vein on day 0 with 10,000 luciferase-positive T47D metastatic breast cancer stem cells mixed with NME7AB to a final concentration of 32 nM. On days 1 and 2, the animals were further injected into the tail vein with 32 nM of NME7AB, showing an increase in metastasis. This is a system to demonstrate the reversal of established metastasis. On day 5, the animals were intravenously injected with 15 mg / kg of 8F9A4A3, also known as 4A3. Here, the antibody was either the mouse sequence, the humanized 4A3 variant 4.8, or the humanized 4A3 variant 12.3. Figure 52A shows control animals injected with phosphate-buffered saline. Figure 52B shows animals treated with anti-NME7AB humanized 4A3 4.8. Figure 52C shows animals treated with anti-NME7AB humanized 4A3 12.3. Figure 52D shows animals treated with mouse 4A3 anti-NME7AB monoclonal antibody. Figure 52E shows a graph of the quantification of tumor volume bioluminescence measurements obtained with the IVIS instrument. Animals were injected with either PBS or anti-NME7 antibody at a dose of 15 mg / kg every three days. Antibody hu4A34.8 indicates that sequences derived from human heavy chain antibody sequence 1.46 (SEQ ID NO: 1102) and light chain sequence 1.6 (SEQ ID NO: 1104) were used. Antibody hu4A312.3 indicates that sequences derived from human heavy chain antibody sequence 4.4 (SEQ ID NO: 1106) and light chain sequence 4.1 (SEQ ID NO: 1108) were used. Note that half of the animals treated with hu4A3 4.8 were given a partial half dose. [Modes for carrying out the invention]

[0046] definition In this application, "a" and "an" are used to refer to both single and multiple objects.

[0047] Where used herein, “about” or “substantially” generally gives leeway from being limited to an exact number. For example, when used in the context of polypeptide sequence length, “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 they have functional activity, such as binding activity.

[0048] As used herein, “combined” administration with one or more additional therapeutic agents includes simultaneous (cooperative) administration and sequential administration in any order.

[0049] As used herein, "amino acid" and "multiple amino acids" all refer to naturally occurring L-α-amino acids. This definition is intended to include norleucine, ornithine, and homocysteine.

[0050] As used herein, the term “amino acid sequence variant” generally refers to a molecule that has several differences in its amino acid sequence compared to a reference (e.g., natural sequence) polypeptide. Amino acid changes may be substitutions, insertions, deletions, or any desired combination of such changes in the natural amino acid sequence.

[0051] A substitutional variant has at least one amino acid residue removed from the native sequence and a different amino acid inserted in its place. The substitution may be a single substitution, where only one amino acid in the molecule is replaced, or it may be a multiple substitution, where two or more amino acids are replaced in the same molecule.

[0052] Amino acid substitutions within a sequence may 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. Neutral polarity 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. Proteins or fragments exhibiting the same or similar biological activity, or derivatives thereof, and derivatives that are differently modified during or after translation, for example, by glycosylation, protein cleavage, or binding to antibody molecules or other cellular ligands, are also within the scope of the present invention.

[0053] An insertion variant is one in which one or more amino acids are inserted directly adjacent to an amino acid at a specific position in the natural amino acid sequence. Directly adjacent to an amino acid means that it is attached to either the α-carboxyl or α-amino functional group of that amino acid.

[0054] A deletion variant is a molecule in which one or more amino acids are removed from the native amino acid sequence. Typically, a deletion variant has one or two amino acid deletions in a specific region of the molecule.

[0055] As used herein, “fragment” or “functional derivative” refers to biologically active amino acid sequence variants and fragments of the polypeptides of the present invention, as well as covalent modifications, including derivatives obtained by reaction with organic derivatizing agents, post-translational modifications, derivatives comprising non-protein polymers, and immunoadhesins.

[0056] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that, at the dosage and concentration used, are nontoxic to cells or mammals to which they are exposed. Often, pharmaceutically acceptable carriers are pH-buffered aqueous solutions. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphates, 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®.

[0057] As used herein, “pharmaceutically acceptable carriers and / or diluents” include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in therapeutic compositions is intended unless any conventional media or agent is incompatible with the active ingredient. Complementary active ingredients may also be incorporated into the composition.

[0058] For ease of administration and uniformity of dosage, formulating parenteral compositions in dose unit formulations is particularly advantageous. As used herein, a dose unit formulation refers to a physically distinct unit suitable as a unit dose for the target to be treated, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect incorporated into the required pharmaceutical carrier. The specifications for the dose unit formulations of the present invention are defined by, and directly depend upon, (a) the specific properties of the active substance and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the field of pharmaceutical technology, such as active substances for the treatment of diseases in living organisms where there is a pathological condition in which the health of the body is compromised.

[0059] The main active ingredient is formulated in an effective amount for convenient and effective administration, along with a suitable pharmaceutically acceptable carrier in the unit dosage form. The unit dosage form contains, for example, an amount of the main active ingredient ranging from 0.5 μg to approximately 2000 mg. Expressed as a percentage, the active compound is typically present in the carrier at approximately 0.5 μg / ml. In the case of compositions containing supplemental active ingredients, the dosage is determined by referring to the usual dose and method of administration of the ingredients.

[0060] As used herein, “vector,” “polynucleotide vector,” “construct,” and “polynucleotide construct” are used interchangeably. The polynucleotide vectors of the present invention may be in any of several forms, which include, but are not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenovirus coat, and DNA wrapped in another virus or virus-like form (such as herpes simplex, and adenostructures such as polyamides).

[0061] In other words, “host cell” includes individual cells or cell cultures that may or may have been recipients of the vector of the present invention. The host cell includes offspring of a single host cell, and the offspring do not necessarily have to be completely identical (morphologically or in total DNA complement) to the original parent cell, whether by natural, accidental, or planned mutation and / or alteration.

[0062] As used herein, “subject” means a vertebrate, preferably a mammal, and more preferably a human.

[0063] As used herein, “mammal” for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and zoo animals, sports animals or pet animals, dogs, cats, cattle, horses, sheep, pigs, etc. Preferably, the mammal is a human.

[0064] As used herein, “treatment” is a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in the severity of the disease, a stable (i.e., non-worsening) state of the disease, delay or slowing of disease progression, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival predicted without treatment. “Treatment” refers to both therapeutic and preventive or protective measures. Persons requiring treatment include those already suffering from the disability, as well as those for whom the disability should be prevented. To “alleviate” a disease means that the severity of the disease state and / or undesirable clinical symptoms are reduced, and / or the progression over time is slowed or prolonged compared to the situation without treatment.

[0065] As used herein, the peptides "A1", "A2", "B1", "B2", and "B3" refer to human NME7 AB This refers to peptides derived from NME7 that are used to produce or select antibodies that bind to human NME1 but do not bind to (or bind very weakly to) it. The peptides used to produce these antibodies are derived from NME7. AB The following is common to both and NME7-X1: 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)

[0066] Further, for clarity, NME7A (with capital letter "A") refers to the subunit A portion of NME7. NME7a (with lowercase letter "a") refers to full-length NME7 separately described herein. And NME7B (with capital letter "B") refers to the subunit B portion of NME7. NME7b (with lowercase letter "b") refers to an NME7 species with a partial deletion of the DM10 region, which is separately described herein.

[0067] As used herein, the term "antibody-like" refers to a molecule that comprises a portion of an antibody, but can be engineered to comprise an antibody portion that is not a naturally occurring antibody 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 T cells such that the body's immune system is directed to attack a specific target protein or cell. Ylanthia® technology consists of an "antibody-like" library that is a collection of synthetic human Fabs screened for binding to a peptide epitope derived from a target protein. The selected Fab regions can then be engineered into a scaffold or framework such that they resemble an antibody.

[0068] As used herein, “effective dose of drug for inhibiting NME family proteins” refers to the effective dose of drug for interfering with the activating interaction between NME family proteins and their corresponding receptors.

[0069] 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.

[0070] When used herein, "MUC1 * The extracellular domain is primarily 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 that cleavage enzyme changes depending on the cell type, tissue type, or time in cell evolution, MUC1 * The exact sequence of the extracellular domain can change at the N-terminus.

[0071] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor, as defined below: GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (Sequence ID 6). In this regard, the "N-number," such as "N-10 PSMGFR" or simply "N-10," "N-15 PSMGFR" or simply "N-15," or "N-20 PSMGFR" or simply "N-20," refers to the number of amino acid residues deleted at the N-terminus of PSMGFR. Similarly, the "C-number," such as "C-10 PSMGFR" or simply "C-10," "C-15 PSMGFR" or simply "C-15," or "C-20 PSMGFR" or simply "C-20," 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 is a wild-type MUC1 in which 20 amino acids are added to the N-terminus of PSMGFR and 27 amino acids are deleted from the C-terminus. * This refers to the peptide fragment.

[0072] When used herein, "MUC1 * The "extracellular domain" refers to the extracellular portion of the MUC1 protein that lacks the tandem repeat domain. In most cases, MUC1 * It is a cleavage product, and its MUC1 * The portion consists of a tandem repeat, a transmembrane domain, and a short extracellular domain lacking a cytoplasmic end. The exact location of cleavage of MUC1 is unknown, probably because it appears to be cleaved by two or more enzymes. * The extracellular domain contains most of the PSMGFR sequence, but may have an additional 10-20 N-terminal amino acids.

[0073] As used herein, “high homology” is considered to mean 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.

[0074] As used herein, “NME family proteins,” or “NME family member proteins,” numbered 1–10, are proteins grouped together because they all possess at least one NDPK (nucleotide diphosphate enzyme) domain. In some cases, the NDPK domain is non-functional in terms of catalyzing the conversion of ATP to ADP. NME proteins were previously known as NM23 proteins, numbered H1 and H2. Recently, as many as 10 NME family proteins have been identified. In this specification, the terms NM23 and NME are interchangeable. In this specification, the terms NME1, NME2, NME5, NME6, NME7, NME8, and NME9 are used to mean both native proteins and NME variants. In some cases, these variants are more soluble, better expressed in E. coli, or more soluble than the native sequence protein. For example, the NME7 used herein has excellent commercial applicability because its variations enable high-yield expression of soluble, properly folded proteins in E. coli. AB This may refer to natural proteins or variants, such as NME7. AB This variant consists primarily of the NME7 A and B domains, but lacks most of the DM10 domain (SEQ ID NO: 39) that is present at the N-terminus of the native protein. Wherever used herein, "NME1" is interchangeable with "NM23-H1". The present invention is also intended not to be limited by the exact sequence of the NME protein. The mutant NME1-S120G, also called NM23-S120G, is used interchangeably throughout this application. The S120G and P96S mutants are preferred due to their directional properties toward dimerization, but are referred to herein as NM23 dimer, NME1 dimer, or dimeric NME1 or dimeric NM23.

[0075] When referred to herein, NME7 is intended to mean natural NME7 having a molecular weight of approximately 42 kDa.

[0076] The "NME7 family" includes full-length NME7, as well as naturally occurring or artificially produced cleavage forms with molecular weights of approximately 30 kDa, 33 kDa, or approximately 25 kDa; NME7b, NME7-X1, NME7 AB , or deletion or partial deletion variants of the DM10 reader sequence (SEQ ID NO: 162), which is amino acids 1-91 of NME7, as represented by SEQ ID NO: 82 or 147, such as recombinant NME7 protein; or variants in which the sequence can be modified to enable efficient expression or variants that enhance yield, solubility, or other properties that make NME7 more effective or commercially promising. "NME7 family" also refers to "NME7 AB It may also contain "like" proteins, which are proteins in the 30-33 kDa range that are expressed in cancer cells.

[0077] As used herein, “agents for maintaining stem cells in a naive state, or for returning primed stem cells to a naive state” refers to proteins, small molecules, or nucleic acids that maintain stem cells, alone or in combination, in a naive state similar to that of cells in the inner cell population of an embryo. Examples include, but are not limited to, human NME1 dimers, bacterial, fungal, yeast, viral, or parasitic NME proteins, particularly human NME proteins, NME1, NME7, NME7-X1, and NME7 AB Examples include nucleic acids such as siRNAs that have high sequence identity with NME6, 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).

[0078] The term "NME7" is used herein. AB "NME7AB" and "MNE-AB" are used interchangeably.

[0079] As used herein, “multipotency-promoting agents” or “agents that revert somatic cells to a stem-like or cancer-like state” refer to proteins, small molecules, or nucleic acids that, alone or in combination, induce or suppress the expression of specific genes such that their genetic signatures are altered to more closely resemble those of stem cells or cancer cells. Examples include, but are not limited to, NME1 dimers, NME7, NME7-X1, and NME7. AB Nucleic acids such as siRNAs that suppress the expression of 2i, 5i; MBD3 or CHD4 or BRD4 or JMJD6; and microbial NME proteins that have high sequence homology to human NME1, NME2, NME5, NME6, NME7, NME8 or NME9, and preferably high sequence homology to the region containing the NDPK domain.

[0080] With respect to a drug referred to as a "small molecule" as used herein, it may be a synthetic chemical molecule or a chemical-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.

[0081] With respect to a drug referred to as a “natural product” as used herein, the molecule in question may be a chemical or biological molecule, as long as it exists in nature.

[0082] As used herein, FGF, FGF-2, or bFGF refer to fibroblast growth factor (Xu RH et al, 2005; Xu C et al, 2005).

[0083] As used herein, “Rho-related kinase inhibitor” may be a small molecule, peptide, or protein (Rath N, et al, 2012). Rho kinase inhibitors are abbreviated here and elsewhere as ROCi, ROCKi, or Ri. The use of specific Rho kinase inhibitors is intended to be illustrative and may be substituted with any other Rho kinase inhibitor.

[0084] As used herein, the terms “cancer stem cells” or “tumor-initiating cells” refer to cancer cells that express levels of genes associated with a more metastatic or more invasive state of cancer. “Cancer stem cells” or “tumor-initiating cells” also refer to cancer cells that, when transplanted into an animal, can induce a tumor with very few cells. Cancer stem cells and tumor-initiating cells are often resistant to chemotherapy drugs.

[0085] As used herein, the terms “stem / cancer,” “cancer-like,” and “stem-like” refer to a state in which a cell acquires 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. Stem-like cells may be somatic cells that have been induced to a less mature state, such as through increased expression of pluripotent genes. Stem-like cells also refer to cells that have undergone some degree of dedifferentiation or are in a metastable state in which they can be transformed into terminal differentiation. Cancer-like cells may be cancer cells that are not yet fully characterized but exhibit the morphology and characteristics of cancer cells, capable of growing independently of a foothold or causing tumors in animals.

[0086] As used herein, “spacers” or “linkers” of different lengths can be incorporated anywhere in a peptide. Spacer bonds are typically amide bonds, but other functional groups are also possible.

[0087] NME, NME7, and the NME7 protein family The inventors found that NME7 and NME7-X1 are highly expressed in early human stem cells and, even more so, in most cancer cells (Figures 17, 18, 19A-19F, 22, 23, 39, 40, 41, 47, and 48). Figure 17 shows a graph of RT-PCR measurements of NME7-X1 expression in a range of human stem cells and cancer cells. Figure 18 shows a graph of RT-PCR measurements of NME7, NME7a, NME7b, and NME7-X1 expression in a range of human stem cells and cancer cells. NME7a is full-length NME7, NME7b has a small deletion of the DM10 domain, and NME7-X1 has all of the DM10 domain and a small deletion of the N-terminus of the first NDPK A domain. Bars labeled NME7 indicate that primers were used to detect both NME7a and NME7b. Figures 19A-19F show Western blot images of the detection of NME7 expression in various cancer cell lines using antibodies generated by immunization with NME7-derived short-chain peptides. Figure 19A shows a Western blot detected using antibody #52 of the present invention, which binds to NME7-derived peptide A1. Figure 19B shows a Western blot detected using antibody #56 of the present invention, which binds to NME7-derived peptide B1. Figure 19C shows a Western blot detected using antibody #61 of the present invention, which binds to NME7-derived peptide B3. Figures 22A-22E show Western blot images of co-immunoprecipitation experiments. T47D breast cancer cell extracts were incubated with an antibody against the cytoplasmic end of MUC1, Ab-5, or a control antibody, IgG, and co-immunoprecipitation was performed. The gels were blotted with two different commercially available anti-NME7 antibodies, B9 (Figure 22A) and CF7 (Figure 22B). Both gels exhibit characteristic NME7 bands at approximately 33 kDa and 30 kDa. Strip the gels and use MUC1 * Antibodies against the extracellular domain of the substance, anti-PSMGFR (Figure 22C and Figure 22D), were used for re-detection. This was observed in seven NME species and MUC1. * However, it shows that they interact. Recombinant NME7 ABand recombinant NME7-X1 were mixed together, developed on a gel, and then detected with an anti-NME7 antibody. Two specific NME7 species that naturally occur in breast cancer cells and bind to MUC1 * that interact with are NME7 AB -like species and NME7-X1 (Figure 22E). Figures 23A to 23C show Western blot photographs of co-immunoprecipitation experiments. Extracts of human induced pluripotent stem cells, iPS7 cells, or embryonic stem cells, HES3 cells were incubated with an antibody against the cytoplasmic terminal of MUC1, Ab-5, or a control antibody, IgG, followed by co-immunoprecipitation. The gel was blotted with a commercially available anti-NME7 antibody B9 (Figure 23A). Both cell types show distinct NME7 bands at approximately 33 kDa and approximately 30 kDa. The gel was stripped and re-probed with an antibody against the extracellular domain of MUC1 * , anti-PSMGFR (Figure 23B). This indicates that the NME7 species and MUC1 * interact with each other. Recombinant NME7-AB and recombinant NME7-X1 were mixed together, developed on a gel, and then detected with an anti-NME7 antibody. Two specific NME7 species that naturally occur in breast cancer cells and interact with MUC1 * are NME7-AB-like species and NME7-X1 (Figure 23C). Figures 39A to 39C show human lung tissue specimens stained with an anti-NME7 antibody that binds to the B3 peptide. This figure shows the absence of NME7 expression in normal tissue, and the increasing expression of NME7 and increased metastasis accompanying tumor malignancy. Figures 40A to 40C show human small intestine tissue specimens stained with an anti-NME7 antibody that binds to the B3 peptide. This figure shows the absence of NME7 expression in normal tissue, and the increasing expression of NME7 and increased metastasis accompanying tumor malignancy. Figures 41A to 41D show human colon tissue specimens stained with an anti-NME7 antibody that binds to the B3 peptide. This figure shows the absence of NME7 expression in normal tissue, and the increasing expression of NME7 and increased metastasis accompanying tumor malignancy. Figures 47 and 48 show immunofluorescence photographs showing that NME7 is secreted by various cancer cell lines and binds to extracellular receptors of those cancer cell lines.

[0088] Furthermore, the inventors have shown that NME7, like NM23-H1, is effective in both stem cells and cancer cells, and in MUC1 * We demonstrated that it binds to and dimerizes the growth factor receptor (Figure 1). Figure 5 shows the sequence alignment of the A and B domains of NME1 and NME7.

[0089] The inventors recently discovered that NME7 is a primitive form of NME1 (NM23-H1) expressed in very early embryonic stem cells. NME7 is either not expressed at all or at very low levels in adult tissues. However, the inventors found that NME7 is expressed at high levels in cancer cells and tissues, and even higher levels in metastatic cancer cells and tissues. A cleaved form of NME7 may be secreted, allowing it to bind to and be activated by extracellular receptors. The inventors 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 blotting detects full-length NME7 in cell lysates, but detects smaller 30-33 kDa NME7 species in their conditional media. Western blots detected with either an antibody that recognizes NME7 or an antibody that recognizes only the DM10 domain indicate that lower molecular weight NME7 species secreted into the culture medium lack the DM10 domain. These data suggest that naturally occurring NME7 species have approximately the same molecular weight as our recombinant NME7. AB Consistent with the idea that they are comparable, both are secreted, and both lack the 91 amino acids in the DM10 domain that can maintain proteins within cells.

[0090] The inventors discovered a novel 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 125 to 376 NME7 amino acids, while recombinant NME7 produced by the inventors... AB, having a molecular weight of about 33 kDa, spans amino acids 92 to 376 and therefore contains 33 additional N-terminal amino acids. NME7b spans amino acids 37 to 376, lacks only the 37 amino acids of the DM10 domain, and is overexpressed in prostate cancer (Figure 18). The present inventors generated human recombinant NME7-X1, which is a naturally occurring "NME7 AB -like" protein, and demonstrate that it is a secreted 30 kDa NME7 species in cancer cells having a molecular weight slightly smaller than that of the naturally occurring approximately 33 kDa NME7 species thought to be the aforementioned protein.

[0091] The present inventors tested a series of cancer cell lines and found that they express NME7 AB , such as NME7-like proteins AB , or cleavable low-molecular-weight species similar to alternative isoforms such as NME7-X1, and NME7 at high levels.

[0092] Contrary to NM23-H1 (also known as NME1), which must be a dimer, NME7 is a monomer that has two binding regions for the MUC1 * extracellular domain. The present inventors generated recombinant human NME7 lacking the DM10 domain and denote it as NME7 AB . A sandwich ELISA binding assay shows that recombinant NME7 AB simultaneously binds to two PSMGFR peptides, in which the MUC1 * extracellular domain contains most or all of the PSMGFR sequence (Figure 1). Nanoparticle binding assays also demonstrated that NME7 can bind to the PSMGFR portion of the MUC1 * extracellular domain and can induce dimerization thereof.

[0093] Agents that neutralize NME7, block its interaction with binding partners, or suppress its expression are promising anticancer therapeutics. Such agents may be antibodies, small molecules, or nucleic acids. They may act directly on NME7, on molecules that control NME7 expression, or on enzymes that cleave NME7 into a cancer-promoting form.

[0094] The inventors have identified recombinant NME7, such as the NM23-H1 dimer. AB We found that monomers can fully support the proliferation of pluripotent human stem cells in the absence of other growth factors, cytokines, or serum. NME7 and MUC1, which essentially contains a PSMGFR sequence. * Competitively suppressing the interaction between extracellular domains induces stem cell differentiation, while NME7 and MUC1 promote stem cell proliferation and inhibit differentiation. * This demonstrates that it is an interaction between the two.

[0095] Next, the inventors further developed NME7 AB NME7 demonstrated that it alone can fully support the proliferation of human cancer cells. AB When added to normal cancer cell growth medium, it stimulates cancer cell growth, especially MUC1-positive and MUC1 * - Stimulated the proliferation of positive cancer cells. MUC1 * Suppression of the interaction between and NME7 inhibited cancer cell proliferation. Anti-MUC1 * MUC1 by Fab * Blocking growth factor receptors strongly suppressed cancer cell proliferation. Similarly, antibodies that bind to NME7 suppressed cancer cell proliferation. In one example of cancer proliferation suppression by anti-NME7 antibodies, polyclonal antibodies were generated by immunizing animals with a portion of NME7 in the range of amino acids 100-376 (Figures 12 and 13). However, the inventors of this invention have found that NME7 AB Antibodies generated from immunization with shorter peptides from NME7-X1, or from MUC1 and MUC1, were also found to suppress cancer growth. In particular, they suppressed cancer growth. * - Suppresses the proliferation of positive cancers. The anti-NME7 antibody of the present invention suppressed the formation of non-adherent "floating" cells that can migrate from the primary tumor and form metastatic tumor spheres (Figures 14, 16, and 29). The anti-NME7 antibody of the present invention suppressed the upregulation of metastasis and stem cell markers, which are now considered characteristic of metastasis (Figures 15, 30, 31, and 32).

[0096] NME7 causes cancer metastasis The inventors of the present invention, NME7 AB We further found that culturing cancer cells in minimal media containing NME7 induces various cancer cells to transform into a more metastatic state. Evidence of this induced metastatic state includes a shift from adherent cell proliferation to non-adherent cell, also known as "suspension" cell proliferation, accompanied in particular by upregulation of certain metastasis markers in suspension cells. AB These transfer markers that are upregulated after culture in NME7 include, but are not limited to, CXCR4, CHD1 (also known as E-cadherin), MUC1, ALDH1, CD44, and pluripotent stem cell markers such as OCT4, SOX2, NANOG, KLF2 / 4, FOXa2, TBX3, ZEB2, and c-Myc (Figures 2, 3, 20, 49, and 51). AB Cancer cells cultured within the medium exhibit an extremely high engraftment rate of over 90% when xenotransplanted into test animals. Furthermore, a very small number of transplanted cancer cells form tumors in the test animals, which is characteristic of NME7. AB This is evidence that they were transformed into cancer stem cells, which are known as metastatic cancer cells. (NME7) AB Cancer cells cultured in and injected into NOD / SCID / GAMMA mice containing estrogen-releasing pellets metastasized from fewer cells in the animals compared to parent cells grown in normal culture medium (Figures 33-38). The cancer cells were cleaved from NME7 cleavage products or NME7 AB Since it produces a selective isoform that is essentially equivalent to NME7, the method described herein is used to produce NME7 AB The use of NME7 is not limited to this, and other NME7 species may function similarly. For example, the inventors have found that another NME7 isoform, NME7-X1, is expressed by cancer cells. It is similar to the inventors' recombinant NME7, except that the X1 isoform lacks 33 amino acids from the N-terminus. AB It is identical to the NME7. The NME7-X1 is the same as the NME7. AB It is expected to function as follows: "NME7 AB "Similar" proteins were also detected in cancer cells, with approximately 33 Da types.

[0097] In their previous work, the inventors of the present invention used NME7 AB The inventors demonstrated that this reagent alone can revert human stem cells to their previous naive state. They also found that culturing cancer cells in the presence of other reagents that revert stem cells to a more naive state transforms the cancer cells into a more metastatic state. The inventors identified NME7 as the solution. AB (Figure 2), or the NME1 dimer (Figure 3), or the "2i" inhibitor (Figure 4) were shown to transform normal cancer cells into metastatic cancer cells called cancer stem cells ("CSCs") or tumor initiation cells ("TICs"), respectively. However, NME7 AB This induced cancer cells to enter a more metastatic state than NME1, also known as NM23-H1, which performed better than 2i.

[0098] 2i is the name given to two biochemical inhibitors that researchers found to restore human stem cells to a more naive state. 2i are the MEK and GSK3 beta inhibitors, PD0325901 and CHIR99021, which are added to the culture medium at final concentrations of approximately 1 mM and 3 mM, respectively. NME7 AB NME7-X1 functions well at lower and higher concentrations, ranging from approximately 1 nM to 16 nM, but is 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, typically at 16 nM in these experiments, and human NME has the S120G mutation. Lower concentrations are required when using the wild type. These exact concentrations are not intended to be critical. Certain mutations tolerate the presence of dimers at higher concentrations, but it is important that the NME1 protein is dimerized, and the concentration range in which this occurs is in the low nanomolar range. Similarly, the concentration of NME7 protein may vary. ABNME7-X1 is a monomer, and the concentration used to transform cancer cells into metastatic cells needs to allow the protein to remain as a monomer.

[0099] NME7, NME7 AB Other researchers have shown that, in addition to the NME7-X1 and 2i inhibitors, MEKi and GSK3i, other reagents and inhibitors can revert stem cells to a more naive state. These inhibitors "i" include JNKi, p38i, PKCi, ROCKi, BMPi, BRAFi, SRCi, as well as the 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 advance cancer cells to a more metastatic state. Cells induced to transform into a more metastatic state using inhibitors or growth factors that revert 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.

[0100] 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 later metastatic cancer cells are assayed by PCR, which measures the expression levels of these genes. The inventors have developed NME7, which transforms them into a more metastatic state, evidenced by increased expression of metastasis markers and pluripotent stem cell markers. AB We demonstrated that these cancer cells, cultured in drugs such as [drug names], function as metastatic cancer cells.

[0101] A functional test to determine whether a population of cancer cells is metastatic involves injecting a very small number of cells, e.g., 200, into immunodeficient mice and observing whether they develop into tumors. Typically, 5 to 6 million cancer cells are required to form a tumor in immunodeficient mice. The inventors have shown that as few as 50 NME-induced metastatic cancer cells formed a tumor in mice. Furthermore, human NME7 AB Mice injected with NME1 or NME7-X1 during the study period developed distant metastases.

[0102] In one specific experiment, T47D human breast cancer cells were cultured in standard RPMI medium for 14 days, with the medium changed every 48 hours, and subjected to trypsin treatment at approximately 75% concentration. The cells were then seeded into 6-well plates and treated with 4nM NME7. AB The cells were cultured in minimal stem cell medium supplemented with (see Example 1). The medium was changed every 48 hours. By approximately day 4, some cells detached from the surface and became suspended. Since these are the cells with the highest metastatic potential, as evidenced by RT-PCR measurement of metastasis markers, the medium was carefully changed to retain the "suspended cells". On day 7 or 8, the suspended cells were collected and counted. The samples were retained for RT-PCR measurement. The important marker measured was CXCR4, which corresponds to NME7 AB After being cultured for a short time, the levels are controlled to increase by 40 to 200 times.

[0103] Newly recovered suspension metastatic cells were xenotransplanted into the flanks of female nu / nu athymoid mice that had been implanted with a 90-day sustained-release estrogen pellet. Suspension cells were xenotransplanted in groups of 10,000, 1,000, 100, or 50 cells, respectively. Half of the mice in each group of six also received 32nM NME7 near the original transplant site. AB NME7 was injected daily. ABParental T47D cells cultured in RPMI medium without NME7 were also transplanted into mice as a control in quantities of 6 million, 10,000, or 100. Mice transplanted with NME7-induced suspension cells developed tumors even when only 50 cells were transplanted. AB Mice that received daily injections of NME7 also developed distant tumors or distant metastases in various organs. AB Human NME7 after transplantation of cultured cancer cells AB Of the 12 mice injected with the drug, 11 (92%) developed tumors at the injection site. After transplantation, human NME7 AB Only 7 out of 12 mice that were not injected with the drug, i.e., 58%, developed tumors. Those showing tumors and human NME7 AB Of the 11 mice injected with NME7, 9 (82%) developed multiple tumors in areas distant from the injection site. AB None of the mice that were not injected developed multiple visible tumors.

[0104] After slaughter, RT-PCR and Western blotting were performed on NME7 AB The distant elevations in the injected mice were indeed human mammary gland tumors. Similar analyses of those organs showed that, in addition to the distant elevations, the mice had randomly metastasized to the liver and lungs, accompanied by the human mammary gland cancer characteristics of the transplanted human mammary gland cancer cells. As expected, only mice injected with 6 million cells developed tumor growth.

[0105] The inventors of this invention have developed a human recombinant NME7 AB The size and arrangement of NME7 were shown to be equivalent to NME7-X1 and 30-33 kDa NME7 cleavage products. The inventors of the present invention have shown that NME7 ABHowever, it was shown that it promotes cancerous growth and accelerates cancer cells to a highly metastatic cancer stem cell (CSC) state, also known as tumor initiation cells (TICs). Therefore, the inventors concluded that NME7-X1 and NME7 cleavage products lacking the DM10 domain also promote cancerous growth and accelerate cancer cells to a highly metastatic cancer stem cell (CSC) state, also known as tumor initiation cells (TICs). In one example, NME7 AB This was added to cancer cells in serum-free medium and under conditions where no other growth factors or cytokines were present. Within 7–10 days, the cancer cells reverted to highly metastatic CSC / TIC, as evidenced by a more than 100-fold increase in the expression of molecular markers such as CXCR4, an indicator of metastatic cancer cells. In one example, T47D breast cancer cells were added to standard RPMI medium or recombinant NME7 at a final concentration of 16 nM. AB The cells were cultured in minimal stem cell medium (Example 1) supplemented with [substance name]. After 10 days, the cells were collected and analyzed by RT-PCR for the expression of molecular markers of CSCs, which had been increased 10 to 200 times (Figure 2). This is a specific and detailed example of how the inventors transformed one type of cancer cell into a more metastatic state. There is a set of cancer cells that are transformed in this way, a set of drugs that return stem cells to a more naive state and further develop cancer cells into a more metastatic state, and a set of concentrations in which the added drugs transform cancer cells, so the invention is not intended to be limited by these details. Other types of cancer cells are NME7 due to the dramatic increase in the expression of metastasis markers and the ability to form tumors from a very small number of transplanted cancer cells. AB A longer incubation period was required in the case of NME7. AB Prostate cancer cells cultured in 2i, human NME1, or bacterial NME1 with high homology to human NME1 or human NME7 showed a dramatic increase in metastasis markers after 2-3 passages.

[0106] 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.

[0107] DU145 prostate cancer cells were cultured similarly, and NME7 AB These cells cultured in NME7 also showed a dramatic increase in CSC marker expression (Figure 3). In prostate cancer cells, CHD1 (also known as E-cadherin) and CXCR4 were found to be present in NME7 AB Compared to control cancer cells that did not proliferate within the cells, pluripotent stem cell growth was upregulated along with other pluripotent stem cell markers. Figures 20A-20C show that all ovarian cancer cell lines SK-OV3, OV-90, and breast cancer cell line MDA-MB transitioned from adherent to non-adherent suspension cells, and NME7 AB This shows that the expression of metastasis markers increased after 72 or 144 hours of culture. Ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, and melanoma cells also showed increased expression of NME7. AB They were cultured in and transformed into a more metastatic state after only 3 days of culture. Figure 21 shows breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and melanoma cells transformed into MUC1 and MUC1 * This indicates that it expresses [the substance].

[0108] Here, the inventors of the present invention have found that NME7 AB However, it was shown that it transforms a wide range of cancer cells into a more metastatic state. The inventors also showed that cancer cells undergo recombinant NME7 AB It has a molecular weight of almost the same as 33 kDa (Figures 17, 18, 19, and 22) and is NME7 AB Naturally occurring species lacking the DM10 domain, and NME7 except for the absence of 33 amino acids from the N-terminus. ABWe demonstrated that the selective isoform NME7-X1 30kDa, which has the same sequence as MUC1, is also expressed. Co-immunoprecipitation experiments were performed on T47D breast cancer cells. Cell extracts were incubated with the cytoplasmic end of MUC1, Ab-5 or a control antibody, or an antibody against IgG, and co-immunoprecipitation was performed. The immunoprecipitated species were separated by gel electrophoresis. The gels were blotted with two different commercially available anti-NME7 antibodies. Both gels showed characteristic NME7 bands at approximately 33kDa and approximately 30kDa (Figures 22A and 22B). The gels were stripped and MUC1 * Antibodies against the extracellular domain of the substance, anti-PSMGFR (Figures 22C and 22D), were used for re-detection. This was observed in seven NME species and MUC1. * However, this shows that they interact. Recombinant NME7 prepared by the inventors. AB The recombinant NME7-X1 was mixed together, spread on a gel, and then detected with an anti-NME7 antibody. It spontaneously occurs in breast cancer cells and MUC1 * Two specific types of NME7 interact with NME7 AB This shows the different species and that they are NME7-X1 (Figure 22E). Similar experiments were performed on human stem cells. Figures 23A-23C show photographs of Western blots from 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 end of MUC1, Ab-5, or a control antibody, IgG, and co-immunoprecipitation was performed. The gel was blotted with commercially available anti-NME7 antibody B9 (Figure 23A). Both cell types show characteristic NME7 bands at approximately 33 kDa and 30 kDa. The gel was stripped and MUC1 * It was re-detected using an antibody against the extracellular domain of NME, anti-PSMGFR (Figure 23B). This was NME7 and MUC1 * This demonstrates that they interact. Recombinant NME7 prepared by the inventors. AB The recombinant NME7-X1 was mixed together, spread on a gel, and then detected with an anti-NME7 antibody. It spontaneously occurs in breast cancer cells and MUC1 * Two specific types of NME7 interact with NME7 AB This indicates the species and that it is NME7-X1 (Figure 23C). NME7AB Since it is a recombinant protein, naturally occurring species may contain an extra 1 to 15 additional amino acids or recombinant NME7 AB It is unclear whether it lacks 1 to 15 additional amino acids, but it develops at the same apparent molecular weight. AB The reason why recombinant NME7 is considered beneficial is that it can stimulate cancer cell proliferation, induce the transition of cancer cells to a more metastatic state, and fully support the development of pluripotency in human stem cells. AB The intention is to develop seven types of NMEs with an apparent molecular weight of approximately 33 kDa that can function as intended.

[0109] The inventors concluded that cancer cell lines and cancer cell populations expressing NME7 and lower molecular weight variants of NME7 include several cancer cells that are CSCs or metastatic cancer cells. These cancers are NME7 AB By culturing cells in NME7-X1, or NME7 species with lower molecular weights, it may be possible to increase the population of cells that are more or are already metastatic. Figure 19 shows all NME7, as well as NME7 with a lower molecular weight of 33 kDa. AB Figure 21 shows Western blots of a series of cancer cells expressing NME7-X1 at 30 kDa. All of the T47D breast cancer cell line, PC3 and DU145 prostate cancer cell line, BT-474 breast cancer cell line, CHL-1 and A2058 melanoma cell line, and CAPAN-2 and PANC-1 pancreatic cancer cell line express MUC1, MUC1 * This shows that the cells express MUC1 or MUC1. In Figure 21A, BT474 cells express MUC1 or MUC1 * Although they do not appear to express MUC1, the inventors previously showed that when these HER2-positive breast cancer cells become resistant to chemotherapy drugs, i.e., metastatic, they express MUC1 * We showed that increasing the expression of anti-MUC1 leads to metastatic properties (Fessler et al 2009) (Figure 21D). * MUC1 by Fab *Receptor blockade reversed their resistance to Herceptin (Figure 21E), Taxol (Figure 21F), and other chemotherapeutic agents. These cancer types and other cancer types express NME7 and lower molecular weight NME7 variants such as 33kDa and 30kDa. AB Culturing cells in NME7-X1 or lower molecular weight NME7 species may make them more transmissible, or increase the number of transmissible cell populations.

[0110] Conversely, the metastatic potential of these and other cancer types expressing NME7 and lower molecular weight NME7 variants such as 33kDa or 30kDa can be reversed by treating the cells with an anti-NME7 antibody. AB Alternatively, an antibody that binds to NME7-X1 is administered to a patient for the treatment or prevention of cancer, including breast, prostate, ovarian, pancreatic, and liver cancer. The inventors of the present invention have identified NME7 AB However, MUC1 * Since it has been shown that anti-NME7 antibodies exert their oncogenic effects by binding to and activating growth factor receptors, they can be used against any MUC1, including but not limited to breast, lung, liver, pancreas, stomach, colorectal, prostate, brain, melanoma, kidney, etc. * It appears to be effective against positive cancers. Anti-NME7, anti-NME7 AB , or an anti-NME7-X1 antibody, NME7 AB NME7 AB Mrs., or NME7-X1 positive, or MUC1 * It is administered to patients for the treatment or prevention of positive cancer.

[0111] Testing of patient cancer cells for effective treatment NPE7 ABNME7-X1 and 2i, along with other reagents that return stem cells to a more naive state, also induce cancer cells to transform into a more metastatic state. After treatment with any one or a combination of these reagents, cancer cells have a higher engraftment rate and require up to 100,000 times fewer cells to induce tumor formation in test animals. Therefore, the methods described herein can be used to enable xenotransplantation of a patient's primary tumor cells into test animals.

[0112] Candidate therapeutic agents can then be tested in recipient animals. Effective therapeutic agents thus identified can be used to treat donor patients or other patients with similar cancers. In one embodiment, a method for identifying effective therapeutic agents for a particular patient or a particular type of cancer includes the following steps: 1) Cancer cells are obtained from a cell line, patient, or patient to whom the investigational therapeutic agent is to be administered; 2) Cancer cells are NME7 AB 3) The resulting cancer cells are cultured in NME7-X1, human NME1, bacterial NME1, 2i, or other reagents that have been shown to revert stem cells to a more naive state; 4) the resulting cancer cells are cultured in human NME7 AB The animals may be transplanted into test animals to which other reagents have been shown to revert stem cells to a more naive state, such as NME7-X1, human NME1, bacterial NME1, 2i with high homology to human NME1 or NME7, or if the animals are human NME7 AB Alternatively, the steps include: 4) the gene is introduced into the animal; 5) the candidate anticancer drug is administered to the animal; 6) the efficacy of the drug is evaluated; and 7) the effective drug is administered to the donor patient or another patient with a similar cancer.

[0113] Anti-NME7 antibody Anti-NME7 antibodies are powerful anticancer agents. NME7 is a growth factor that promotes the proliferation of cancer cells and further accelerates their progression to a more metastatic or higher-grade state. NME7, and cleaved forms of NME7 at approximately 33 kDa or 30 kDa, have been shown to fully support cancer growth even in serum-free media lacking any other growth factors or cytokines. In pull-down assays, ELISA, and nanoparticle binding experiments, we have shown that growth factor receptor MUC1 * NME7 and NME7 AB It was shown to be a binding partner. Promoting this interaction by eliminating all other growth factors or cytokines increased the expression of cancer stem cell markers. Blocking the interaction using a polyclonal antibody that specifically binds to NME7 actively killed cancer cells even in the presence of serum. Therefore, anti-NME7 or anti-NME7 AB Antibodies are powerful anticancer agents that can be administered to patients for the treatment or prevention of cancer. More than 75% of all cancers are caused by MUC1 * Positive. MUC1 * This is a transmembrane cleavage product of MUC1, in which most of the extracellular domain is deleted, leaving a portion of the extracellular domain containing most of the PSMGFR sequence, and may contain 9 to 20 additional amino acids at the N-terminus of the PSMGFR sequence boundary.

[0114] One aspect of the present invention is a method for treating or preventing cancer in a subject, comprising administering an effective amount of anti-NME7 antibody to the subject. In one example, the anti-NME7 antibody is NME7 AB It can bind to. In another example, an anti-NME7 antibody can bind to NME7-X1. In yet another example, an anti-NME7 antibody administered to a patient inhibits or prevents its binding to its target in promoting cancer. In one example, the target is the extracellular domain of cleaved MUC1. More specifically, the NME7 target that promotes cancer is MUC1 * This is the PSMGFR region of the extracellular domain. In one embodiment, effective therapeutic agents are primarily MUC1 * Seven types of NMEs consisting of the PSMGFR moiety or PSMGFR peptide, and MUC1 *These are drugs that disrupt or prevent interactions with the extracellular domain. Drugs used for the treatment or prevention of cancer include NME7, NME7 AB These are drugs that directly or indirectly inhibit the expression or function of selective isoforms containing NME7-X1 or similar cleavage products. For example, effective anticancer drugs are those that bind to or inactivate the tumor activity of NME7 species. Effective therapeutic agents for the treatment or prevention of cancer include NME7, NME7 AB The therapeutic agent is a cleavage product, or a selective isoform, or a drug that binds to or disables NME7-X1. In one embodiment, the therapeutic agent that binds to an NME7 species is an antibody. The antibody may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or an antibody mimetic, human animal chimera, humanized, or human antibody, of which the origin may be animal. The antibody can be produced by inoculation or immunization with an NME7 species or a fragment thereof, or, for example, from an antibody library or antibody pool. AB Selective isoforms containing cleavage products or NME7-X1 can be chosen based on their binding ability to NME7 species.

[0115] Production of anti-NME7 antibodies Anti-NME7 antibodies can be produced outside the patient, such as in a host animal, or within the patient. Antibodies can be produced by immunization with NME7 or NME7 fragments, or by NME7 AB Alternatively, antibodies can be selected from a library or pool of antibodies, which may be natural, synthetic, full-length antibodies or antibody fragments, based on their binding ability to a desired NME7 species, such as NME7-X1. In one embodiment, antibodies are generated from immunization with peptides selected from those listed in Figures 6-9, or are selected for their binding ability to peptides. In another embodiment, antibodies are generated from peptides whose sequences are not identical to those of human NME1, or antibodies are selected for their binding ability to NME7 species and their inability to bind to human NME1.

[0116] One method used to identify NME7 or NME7-X1-derived peptides, or peptides that are themselves inhibitory, that produce antibodies that suppress cancer growth and inhibit metastasis is as follows: 1) Protein sequences of human NME1, human NME7, human NME7-X1, and several bacterial or fungal NME proteins that have high sequence homology to human NME1 or human NME7 are aligned; 2) Regions with high sequence homology in all NMEs are identified; 3) Peptide sequences specific to NME7 or NME7-X1 but adjacent to regions with high sequence homology are identified. The peptides are then synthesized and used to produce antibodies in humans or host animals. The resulting antibodies are selected for therapeutic use in the following cases: 1) They are NME7 AB 1) It binds to NME7-X1 but not to NME1; 2) It has the ability to suppress cancer growth; 3) It has the ability to suppress the migration of cancer cells to a more metastatic state; or 4) It suppresses metastasis in vivo. In some cases, antibodies for therapeutic use are NME7 AB Or MUC1 of NME7-X1 * They are selected due to their ability to cleave binding to extracellular domains, PSMGFR peptides, or N-10 peptides.

[0117] Use of anti-NME7 antibodies for cancer treatment Antibodies that inhibit cancer growth or the transition to a more metastatic state are selected for use as anticancer drugs and can be administered to patients for the treatment or prevention of cancer. 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, the inventors selected NME7 peptides from the NME7 region, which they believe to be essential to its oncogenic function. The inventors then used these peptides to produce antibodies and subsequently tested both the resulting antibodies and the immunized peptides for their ability to a) inhibit cancer growth; and b) inhibit the induction of the transition from cancer cells to metastatic cancer cells. NME7 peptides were selected as both an immunizing agent for antibody production and as an inhibitor itself (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 represents the domain from which the peptide originates, i.e., the NDPK A domain, and B represents the NDPK B domain from which the peptide originates (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 blood of the immunized rabbits were purified using a column derivatized with the immunizing peptides. The purified antibodies were then tested for their binding ability to human NME7. All obtained antibodies bound to human NME7 as desired, but not to human NME1 (Figures 10A-10B, Example 8). These results indicate that by selecting peptides whose sequences are found in NME7 but are not exactly identical to those in NME1, antibodies that specifically bind to NME7 but not to NME1 can be produced. Since NME1 has healthy function, it is almost always desirable to produce antibodies that do not interfere with NME1. Antibodies also work with MUC1. * Their ability to inhibit the binding of NME7 to extracellular domain peptides was tested. ELISA experiments shown in Figure 11 showed that the antibodies inhibited the binding of MUC1 far more than they inhibited the binding of NME1. * NME7 to extracellular domain peptides ABThis indicates that the binding was inhibited. Recall that the respective NME7 A domain and B domain can bind to the PSMGFR peptide. Therefore, NME7 AB Complete inhibition of binding to the PSMGFR peptide cannot be achieved with a single antibody or a peptide derived from only one domain. These antibodies and their respective immunized peptides also suppressed cancer cell proliferation (Figures 12-13). These antibodies also suppressed NME7 AB The formation of non-adherent "floating" cells arising from cancer cells proliferating within the cells was suppressed (Figure 14). As can be seen from the figure, the polyclonal antibody purified by immunization with B3 peptide reduced the number of metastatic floating cells by 95%, indicating that the anti-NME7 antibody bound to B3 peptide is the most effective in suppressing cancer metastasis. Similarly, the antibody suppressed the expression of the metastasis marker CXCR4 (Figure 15A). In this case as well, the B3 antibody was the most efficient in suppressing CXCR4 expression; the bar labeled NME7 FL (NME7 floating cells) shows a 20-fold reduction with B3 antibody 61 (bar labeled NME7+61FL), and a 70-fold increase in CXCR4. In addition, the immunized peptide itself showed that T47D cancer cells were affected by NME7 AB Alternatively, if proliferation occurred in 2i, it suppressed the upregulation of CXCR4 and other metastasis markers.

[0118] This is just one example of a selective peptide that generates antibodies to suppress the oncological functions of NME7 and NME7 species. Sequence alignment between human NME1, human NME7, human NME7-X1, and bacterial NME proteins with 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 cancerous growth or metastatic states suggests that the five regions from which these peptides originate are critical regions of NME7 for its function in promoting cancer. Other peptides from these regions would also likely suppress cancer growth and metastasis, thus giving rise to anti-NME7 antibodies, which are powerful anticancer drugs. Antibodies generated from peptides A1, A2, B1, B2, and B3 showed inhibition of cancer growth and suppression of the transition to more metastatic states. Monoclonal antibodies produced by immunization with the same or similar peptides and subsequent testing of the monoclonal antibodies will identify antibodies that, after humanization or other manipulations known to those skilled in the art, may be administered to patients for the treatment or prevention of cancer.

[0119] In specific experiments, antibodies produced by immunization with peptides A1, A2, B1, B2, and B3, as well as the immunizing peptides themselves, were added to cultured cancer cells to determine whether the addition of antibodies or immunizing peptides inhibited cancer cell proliferation. At low concentrations and added separately, the antibodies and immunizing peptides suppressed cancer cell proliferation (Figure 12 for one example). However, at higher concentrations or added in combination, the antibodies and immunizing peptides strongly inhibited cancer cell proliferation (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, and 343-371, respectively, from the human full-length NME7 having SEQ ID NOs. 82 or 147.

[0120] To clarify, when NME7 residue numbers are discussed, they refer to the residue numbers of NME7 shown in SEQ ID NOs. 82 or 147.

[0121] The antibodies used in the cancer growth suppression experiments and one of the antibodies shown in Figure 12 were generated by immunization 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 taken: immunization of animals with a peptide containing human NME7 amino acids 100-376, and then: 1) removing the antibody that binds to human NME1; 2) removing the NME7 AB 1) Select an antibody that suppresses other NMEs that induce the transition of cancer cells to a more metastatic state; 2) Select an antibody that suppresses the proliferation of cancer cells; 3) Select an antibody that suppresses MUC1 * Select an antibody that suppresses the proliferation of positive cancer cells; 5) MUC1 * NME7 to the extracellular domain AB Alternatively, select an antibody that inhibits the binding of NME7-X1, or substantially inhibits its binding to the PSMGFR peptide; and / or 6) select an antibody that binds to one or more of the peptides A1, A2, B1, B2, or B3 listed in Figure 9.

[0122] Monoclonal antibodies with higher affinity, or monoclonal antibodies derived from longer peptides, may be more effective antibody therapies. Alternatively, anti-NME7, anti-NME7 AB Alternatively, a combination of anti-NME7-X1 antibodies may be administered to the patient to enhance efficacy.

[0123] Anti-NME7 antibodies suppress the migration of cancer cells to metastatic cancer cells. The anti-NME7 antibody suppresses the migration of cancer cells to metastatic cancer cells or tumor initiation cells (TICs), also known as cancer stem cells (CSCs). AB It should be recalled that culturing various cancer cells in the presence of [NME7] demonstrated that these cancer cells could be transformed from normal cancer cells into metastatic CSCs or TICs. Therefore, NME7, NME7 AB Alternatively, antibodies that bind to NME7-X1 may suppress the progression of cancer cells to a more metastatic state.

[0124] Cancer cells transform into a more metastatic state when cultured in the presence of a drug that reverts stem cells to a more naive state. The inventors of NME7 AB We demonstrated that culturing cancer cells in human NME1 dimers, bacterial NME1 dimers, 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 hypoadherent and float away from the culture dish. These floating cells, "floats," were collected separately from the adherent ones and were shown to: a) express much higher levels of metastatic genes; and b) generate tumors when xenotransplanted 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, and KLF4 were found to be NME7 AB It was dramatically overexpressed in cancer cells cultured within the medium, and most overexpressed in non-adherent cells, referred to here and in the figure as "suspends."

[0125] In one embodiment, NME7 was produced by immunization with NME7-derived peptides A1, A2, B1, B2, and B3. AB To determine whether specific antibodies, as well as immunizing peptides themselves, suppress the transformation of normal cancer cells into metastatic cancer stem cells, NME7 AB Alternatively, it was added to the culture medium together with either of 2i. The antibody and peptide were added separately to the drug that induces translocation transformation, in this case NME7. AB Alternatively, it was added together with the 2i inhibitors PD0325901 and CHIR99021. NME7 AB and 2i were used separately to induce cancer cells to transform into a more malignant metastatic state. In 2i, the antibody added to the culture medium simply did not affect all NME7 AB It was used to block the opening, so that it could not be claimed that the causative agent was not effectively present (Example 10).

[0126] Visual observations were independently recorded by two scientists as the experiment progressed (Figure 14). The most striking observation was that the antibody and peptide dramatically reduced the number of suspension cells, which was the first indicator that the antibody and peptide suppressed the transformation into metastatic cancer cells. In particular, cells produced from immunization with the antibody and B3 peptide produced almost no suspension cells. mRNA was extracted from both suspension cells, adherent cells, and control cancer cells. The amount of mRNA indicating cell viability 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 This study confirms that the antibody suppresses cancer cell proliferation and their transition to a more metastatic state. Expression levels of metastasis markers, including CXCR4, were measured using RT-PCR. Treatment with anti-NME7 antibody significantly reduced the levels 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 support the NME7 AB This demonstrates that antibodies that bind to [specific substance] can be administered to patients for the treatment or prevention of metastatic cancer.

[0127] NPE7 AB , or peptides derived from NME7-X1, intact NME7 AB It competitively inhibits the binding of NME7-X1 and is also an anticancer agent. In another aspect of the present 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, the NME7-derived peptide is administered to a patient, thereby the peptide, which is shorter than the complete NME7 and should not be able to impart the oncogenic activity of NME7, binds to the target of NME7 and competitively suppresses the cancer-promoting interaction between the target and intact NME7. AB Since it can completely confer carcinogenic activity, NME7 ABThe sequence is preferred as a shorter peptide source, in which case it is necessary to ensure that the peptide itself does not promote cancer growth or other tumor or carcinogenic activity. In a preferred embodiment, NME7 AB One or more peptides having a partial sequence, preferably with an amino acid length of about 12 to 56, are administered to the patient. To increase the half-life, the peptide may be a peptide mimetic, such as a peptide with a non-natural skeleton or a peptide with D-type amino acids instead of L-type amino acids. In another example, the anticancer drug is a peptide or a peptide mimetic, and the peptide is NME7, NME7 AB MUC1, or NME7-X1, or its target, the PSMGFR peptide, also known as "FLR" in some instances herein. * It has a sequence that is highly homologous to at least a portion of the extracellular domain.

[0128] Figures 6–9 provide a list of preferred amino acid sequences expected to inhibit the binding of NME7 to its congener target. In a more preferred embodiment, peptides selected for administration to patients with cancer or at risk of developing cancer are selected because they bind to an NME7 binding partner and the peptide itself does not derive tumor activity. In a more preferred embodiment, the NME7 binding partner is MUC1 * This is the extracellular domain. In a more preferred embodiment, the NME7 binding partner is the PSMGFR peptide.

[0129] The term "contributes tumor activity or carcinogenic activity" means that the peptide itself cannot support or promote cancer growth. Another way to test whether peptides or multiple peptides derived from NME7 can promote tumorigenesis is to test whether the peptides can support the pluripotent proliferation of human stem cells. NME proteins and peptides that support the proliferation of pluripotent human stem cells also support cancer growth. Another way is to deselect peptides if they can revert somatic cells to a lower maturity state.

[0130] NPE7 AB The fragments suppress cancer cell proliferation and the transition of cancer cells to a more metastatic state. For example, NME7 peptides A1, A2, B1, B2, and B3, added separately (Figure 12) or in combination (Figure 13), suppressed cancer cell proliferation. Furthermore, NME7 peptides A1, A2, B1, B2, and B3 suppressed the transition of cancer cells to a more metastatic state (Figure 15).

[0131] Therefore, specific to NME7, and NME7 AB Alternatively, antibodies produced by immunization with peptides specific to NME7-X1 may inhibit the carcinogenic effects of NME7 species and could be powerful anticancer agents. Similarly, these results suggest that antibodies specific to NME7, and NME7 AB Alternatively, peptides specific to NME7-X1 inhibit the carcinogenic effects of NME7 species. In one aspect of the present invention, the peptide is selected from the list shown in Figure 6. In one aspect of the present invention, the peptide is selected from the list shown in Figure 7. In one aspect of the present invention, the peptide is selected from the list shown in Figure 8. In yet another aspect of the present invention, the peptide is selected from the list shown in Figure 9. These antibodies may be produced by immunization, or produced or selected by other means, and then, without limitation, contain 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) in NME7, NME7 AB They are selected for their ability to bind to NME7-X1 or NME7-derived peptides. Such antibodies may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, human, or humanized antibodies, or antibody mimics such as protein scaffolds that provide a recognition region for binding to a specific target.

[0132] Anti-NME7 antibodies used in the treatment or prevention of cancer can be prepared by standard methods known to those skilled in the art, and these methods involve NME7, NME7 ABAlternatively, NME7 lacks the additional 10-25 amino acids from the N-terminus. AB These are used to generate antibodies or antibody-like molecules that recognize shorter forms of [substance name]. Such antibodies may be human antibodies or humanized antibodies. Such antibodies may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, human, or humanized antibodies, or may be antibody mimics such as protein scaffolds that provide a recognition region that binds to a specific target.

[0133] Anti-NME7 antibodies produced 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 peptides A1, A2, B1, B2, or B3, are NME7 AB It is an antibody that binds to NME7-X1 but resists binding to NME1, which may be necessary for the function of some healthy cells. Such an antibody is NME7 AB Or their target receptors for NME7-X1, MUC1 * It inhibits binding to the A1, A2, B1, B2, or B3 peptides. 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 may be human antibodies or humanized antibodies. Such antibodies may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or antibody mimics such as protein scaffolds that provide a recognition region for binding to a specific target.

[0134] Anti-NME7 antibodies produced by immunization with B3 peptide, or antibodies that bind to B3 peptide, are NME7 AB And it is particularly specific with respect to the recognition of NME7-X1. Such antibodies are NME7 AB Or their target receptors for NME7-X1, MUC1 *They are also highly efficient in inhibiting binding to the B3 peptide. Antibodies that bind to the B3 peptide are also highly efficient in preventing, suppressing, and reversing cancer or cancer metastasis. Such antibodies may be human antibodies or humanized antibodies. Such antibodies may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or antibody mimics such as protein scaffolds that provide a recognition region for binding to a specific target.

[0135] It should be noted that polyclonal antibody #61, produced by immunization with B3 peptide in rabbits, suppressed the transformation of cancer cells into cancer stem cells, as evidenced by antibody #61, which blocks the increased expression of the transfer marker CXCR4 (Figure 15).

[0136] The B3 peptide derived from NME7 (SEQ ID NO: 145) has a cysteine ​​at position 14, which complicates the production of anti-NME7 antibodies. The inventors mutated cysteine ​​14 to serine to create AIFGKTKIQNAVHSTDLPEDGLLEVQYFF (SEQ ID NO: 169), immunized animals with this antibody, and produced anti-NME7 monoclonal antibodies. The resulting antibodies bind to the native B3 sequence and the B3Cys14Ser peptide. Seven high-affinity and specific monoclonal antibodies were produced: 8F9A5A1, 8F9A4A3, 5F3A5D4, 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4. However, various sequence alignments have shown that only three specific sequence antibodies exist: 8F9A5A1, 8F9A4A3, 8F9A4P3, and 5F3A5D4, as shown below. The regions shown in bold and italics represent the CDR sequences.

[0137] Heavy chain alignment [ka] JPEG2026143419000002.jpg92155

[0138] Light chain alignment [ka] TIFF2026143419000004.tif47152 JPEG2026143419000005.jpg27153

[0139] The monoclonal antibodies 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4 all have the same sequence as 5F3A5D4, also known as 5D4. Here, when the inventors refer to antibody 5F3A5D4, also known as 5D4, it is understood that this also applies to 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4. As can be seen from Figures 24 and 25, the anti-NME7 antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4 all have the same sequence as NME7 AB It binds to NME7 but not 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 anticancer drugs or anti-metastatic drugs, NME7 AB It will be essential to suppress [the other substance] without suppressing NME1.

[0140] Figures 26, 27, and 28 show that these anti-NME7 antibodies also affect NME7 AB MUC1 * This demonstrates that binding to PSMGFR peptides and N-10 PSMGFR peptides can be cleaved. As can be seen in the figure, MUC1 * NME7 from peptides AB There is no overall substitution for it. However, NME7 AB It consists of domain A and domain B, each of which is MUC1 * It should be noted that these antibodies can bind to the B domain of MUC1. * Designed to break the connection to; NME7 AB The A domain is still on the plate surface MUC1 * It will be able to bind to peptides. For useful therapeutic purposes, antibodies will have a single domain of MUC1.* It is sufficient to simply break the binding to MUC1, thereby enabling ligand-induced dimerization and MUC1 * Activation of growth factor receptors may be inhibited. Antibodies or antibody mimes that bind to NME7 B3 peptide or B3Cys14Ser peptide (SEQ ID NO: 169) are antibodies that can be administered to patients diagnosed with cancer or metastasis, or patients at risk of developing cancer or metastasis.

[0141] It is well known in this field that it is difficult to create metastatic cancer cells in animal models. In human tumors, it is estimated that only 1 in 100,000 cancer cells, or 1 in 1,000,000 cancer cells, will isolate from the tumor, implant somewhere, and initiate metastasis [Al-Hajj et al., 2003]. Some researchers have reported that T47D breast cancer cells injected into immunocompromised mice 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].

[0142] Here, the inventors have shown that T47D breast cancer cells are recombinant NME7 AB This shows that the cells grew for 10 days in serum-free medium containing as the sole growth factor. NME7 AB When grown within the environment, approximately 25% of the cancer cells began to float and ceased division, but remained viable. PCR measurements showed that these "floating" cells significantly upregulated the expression of the breast cancer metastasis factor CXCR4.

[0143] In some of the figures provided herein, these suspended cells are referred to as cancer stem cells (CSCs). Female nu / nu mice with impaired immune function were transplanted with an estrogen-releasing pellet for 90 days. 500,000 T47D-wt cells or 10,000 T47D-CSCs (cancer stem cells) were injected subcutaneously (sc) into the tail vein (i.vi) or into the intraperitoneal space (ip) of the nu / nu mice. These cancer cells were engineered to express luciferase. To visualize tumors or cancer cells, the animals were injected with luciferin and visualized 10 minutes later using an IVIS analyzer. As can be seen from the IVIS measurements in Figures 33A–33B, by day 6, the 500,000 T47D-wt cells injected into the tail vein showed no signs of viable cancer cells or cell engraftment.

[0144] In stark contrast, 10,000 T47D-CSCs injected into the tail vein metastasized. Before the IVIS measurement on day 6, T47D-CSC mice were given 32 nM recombinant NME7. AB The following day, one of the 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. AB The near-simultaneous injection of anti-NME7 antibodies likely counteracted the antibody's effect. Figure 34 shows that by day 10, the treated mice were almost completely metastatic. As can be seen from the figure, the mice selected for treatment were more metastatic than comparable T47D-CSC mice.

[0145] The animals were injected with anti-NME7 antibody again on day 10. IVIS measurements on day 12 (Figure 35) show that antibody-treated mice began to eliminate metastases. By day 14 (Figure 36), untreated mice had died due to severe metastases, while treated mice had eliminated metastases. Figure 37 shows the time course of IVIS measurements for mice injected with 500,000 T47D-wt cells and mice injected with T47D-CSCs, who received anti-NME7 treatment up to day 17, in which case antibody treatment would have been discontinued. As can be seen from the figure, on day 17, small clusters of cancer cells remained, which grew larger by day 19. By day 21, metastases had propagated, and antibody treatment was resumed. 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.

[0146] Figure 38 shows the time course of IVIS in animals injected subcutaneously or intraperitoneally. Antibody infusion into animals injected subcutaneously or intraperitoneally with CSC was also performed with anti-NME7 antibodies by sc or ip. In these animals, antibody infusion was stopped on day 17 and not resumed. Figures 39 and 40 show that polyclonal anti-NME7 antibodies produced by immunization with the B3 peptide strain promoted the progression of advanced and metastatic cancers, but not normal tissue or low-grade cancers, with one in 100,000 cancer cells or one in 1,000,000 cancer cells likely to be metastatic cancer cells. In summary, these data suggest that the anti-NME7 antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4, or 8F9A5A1, 8F9A4A3, or 5F3A5D4 administered to patients diagnosed with cancer or at risk of developing cancer, may prevent, suppress, or reverse the formation of cancer metastases.

[0147] In addition to treating metastatic animals with an anti-NME7AB antibody cocktail, the inventors also demonstrated that monoclonal anti-NME7AB antibodies administered individually can prevent and reverse cancer metastasis. In one demonstration study, approximately 20 g each of 8-10 week old female nu / nu mice were transplanted with estrogen-releasing pellets for 90 days. Cancer cells were affected by the growth factor NME7 AB Metastatic cells were made by culturing them in serum-free medium supplemented with 10–15 days. Both adherent and suspension cells showed upregulation of the metastasis marker and could metastasize in animals within 4–7 days. In this case, suspension cells were harvested on day 11 of in vitro culture and injected into the tail vein of the test animals. To test a prophylactic model, anti-NME7 was injected into the tail vein of one group of animals. AB The antibody 8F9A4A3 was injected at a dose of 15 mg / kg 24 hours before the injection of metastatic cancer cells, and the same dose was then injected approximately every 48 hours thereafter. Figures 42A-42F show 10,000 luciferase-positive T47D metastatic breast cancer stem cells injected into the tail vein, and anti-NME7 AB The images show female nu / nu mice treated with antibody 4A3 (also known as 8F9A4A3). To image cancer cells, luciferin, a luciferase substrate, is intraperitoneally injected 10 minutes before imaging with an IVIS analyzer. Figures 42A–42C show IVIS images of animals facing downwards. Figures 42D–42F show IVIS images of animals facing upwards. Figures 42A and 42D show control animals injected with phosphate-buffered saline. Figures 42B and 42E show animals injected with anti-NME7 24 hours before injection of metastatic cancer cells. AB This shows a prophylactic model in which the animal was injected with antibody 4A3, followed by a total of 12 antibody injections over 22 days, approximately every other day. Figures 42C and 42F show the animal's reaction to anti-NME7 24 hours after injection of metastatic cancer cells. AB This shows a reversed model in which antibody 4A3 was injected, followed by a total of 11 antibody injections over 20 days, approximately every other day. As can be seen from the figure, anti-NME7 AB The antibody 8F9A4A3 can prevent and reverse established metastases.

[0148] Anti-NME7AB 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, and anti-NME7 AB These are photographs of female nu / nu mice weighing approximately 20g each, treated with antibodies 5A1 (also known as 8F9A5A1) and 5D4 (also known as 5F3A5D4). To image cancer cells, the luciferase substrate luciferin was intraperitoneally injected 10 minutes before imaging with an IVIS analyzer. Figures 43A–43C show IVIS images of animals facing downwards. Figures 43D–43F show IVIS images of animals facing upwards. Figures 43A and 43D show control animals injected with phosphate-buffered saline. Figures 43B, 43E, 43C and 43F show animals injected with 15 mg / kg of anti-NME7 24 hours prior to injection of metastatic cancer cells. AB This shows a preventative model in which mice were injected with an antibody, and then received a total of 12 antibody injections approximately every other day over 22 days. The photographs were taken on day 24 or day 27. Specifically, mouse #1, treated with antibody 5A1, was photographed on day 27, while mice #2 and #3 were photographed on day 24 because the animals died on day 26.

[0149] anti-NME7 AB Antibodies 5A1 and 5D4 were also tested in a metastasis reversal model and were shown to significantly suppress established cancer metastases. In this experiment, animals were given NME7 at a final concentration of 32 nM on day 0. AB 10,000 T47D metastatic cancer cells mixed with [a certain substance] were injected into the tail vein. Furthermore, the animals were given more NME7 cells on days 3 and 4, which in our experiments showed formed metastases that were more difficult to reverse. AB The antibody was injected. The first antibody injection was on day 7. Since the degree of metastasis varied somewhat in each test animal, the inventors determined that the apparent removal of metastatic cancer cells was due to anti-NME7 AB It was necessary to confirm that the effect was due to treatment. Therefore, the inventors treated animals using alternating high and low doses. As can be easily seen from Figure 44, high dose anti-NME7AB This resulted in the removal of metastases, and if complete removal was not possible, the treatment was reversed and increased using a lower dose. This experiment tested all three anti-NME7 agents tested. AB The antibodies 5A1, 4A3, and 5D4 can bind to NME7-B3 and suppress cancer metastasis in a concentration-dependent manner. Figures 44A-44D show the binding of NME7 to a final concentration of 32 nM. AB This is a photograph of a female nu / nu mouse into which 10,000 luciferase-positive T47D metastatic breast cancer stem cells mixed with 32 nM NME7 were injected into the tail vein. The animal was then given 32 nM NME7 AB After being injected into the tail vein, individual anti-NME7 AB Treated with antibodies. Figure 44A shows control animals injected with phosphate-buffered saline. Figure 44B shows anti-NME7 AB This shows animals treated with the monoclonal antibody 8F9A5A1. Figure 44C shows anti-NME7 AB This shows animals treated with the monoclonal antibody 8F9A4A3. Figure 44D shows anti-NME7 AB This figure shows animals treated with the monoclonal antibody 5F3A5D4. Green arrows indicate resistance doses (5-7 mg / kg) over the indicated period, and red arrows indicate high doses (15 mg / kg). As can be seen from the figure, metastases largely disappear when the antibody is administered at 15 mg / kg.

[0150] This invention is anti-NME7 AB In addition to demonstrating that antibodies can suppress metastasis, the inventors tested their effects on metastasis from primary tumors. This would more closely mimic the physiology of cancer metastasis. The inventors used T47D metastatic breast cancer cells, also known as cancer stem cells (CSCs), against NME7 ABCancer cells were generated by culturing them in serum-free minimal medium for 10–15 days. These T47D CSCs were then subcutaneously transplanted into the right flank of NSG mice that had been implanted with an estrogen-releasing pellet 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 measured and located by imaging with an IVIS detector. Figures 45A and 45B show NME7 up to a final concentration of 32 nM on day 0. AB The image shows female nu / nu mice that were subcutaneously injected into the right flank with 10,000 luciferase-positive T47D metastatic breast cancer stem cells, which were mixed with Matrigel in a 1:1 vol:vol ratio after being mixed with other substances. Tumor engraftment was allowed to progress from day 0 to day 6. The animals were then treated with anti-NME7. AB IV treatment was administered by tail vein infusion of antibodies. Control animals were infused with PBS. Figure 45A shows an IVIS photograph of a control animal. Figure 45B shows an anti-NME7 AB The image shows an IVIS (Intravenous Vein Injection Situation) photograph of an animal that received a cocktail of antibodies 5A1, 4A3, and 5D4 with a total concentration of 15 mg / kg via tail vein injection. The antibodies or PBS were administered four times between days 7 and 18. As can be seen from the figure, anti-NME7 AB Animals treated with antibodies showed fewer metastases (blue dots throughout the body) than the control group. Within the treatment group, 2 out of 5 animals had primary tumors larger than those in the control group. This is likely due to anti-NME7. AB It is thought that the antibodies hindered the spread of cancer cells, and therefore remained concentrated in the primary tumor. In this experiment, PCR analysis performed before the injection of cancer cells showed NME7 AB After 11 days in cultures containing [the specified substance], T47D breast cancer cells showed upregulation of CXCR4 by 109-fold, OCT4 by 2-fold, NANOG by 3.5-fold, and MUC1 by 2.7-fold.

[0151] In another experiment, the inventors demonstrated that the anti-NME7 of the present invention was effective against metastasis from the primary tumor to organs to which breast cancer typically metastasizes. AB The effectiveness of the antibody was tested. Breast cancer typically metastasizes to the liver, lungs, bones, and brain, in that order. The inventors tested T47D metastatic breast cancer cells against NME7AB These T47D CSCs were generated by culturing cancer cells in serum-free minimal medium for 11 days. These T47D CSCs were then subcutaneously implanted into the right flank of NSG mice that had been implanted with an estrogen-releasing pellet for 90 days. Figures 46A–46P show the NME7 at a final concentration of 32 nM on day 0. AB The image shows female nu / nu mice that were subcutaneously injected into the right flank with 10,000 luciferase-positive T47D metastatic breast cancer stem cells, which were mixed with Matrigel in a 1:1 vol:vol ratio after being mixed with other substances. Tumor engraftment was allowed to progress from day 0 to day 6. The animals were then treated with anti-NME7. AB Antibody was administered via tail vein infusion for intravenous therapy (IV). Control animals were infused with PBS. On day 38, the animals were sacrificed, and their livers were harvested and analyzed with IVIS to detect cancer cells that had metastasized to the liver. Figures 46A and 46B show whole-body IVIS images of control animals infused with PBS alone. Figures 46C and 46D show anti-NME7 AB Whole-body IVIS images of control animals injected with antibody 5A1 are shown. Figures 46E and 46F show anti-NME7 AB Whole-body IVIS images of control animals injected with antibody 4A3 are shown. Figures 46G and 46H show anti-NME7 AB Whole-body IVIS images of control animals injected with antibody 5D4 are shown. Figures 46A, 46C, 46E, and 46G are IVIS images taken 7 days prior to all treatments. Figures 46B, 46D, 46F, and 46H are anti-NME7 images. AB These are IVIS images taken 31 days after antibody treatment or mock treatment. As can be seen from the figure, animals in the PBS control group showed metastasis in whole-body IVIS images (blue dots), while anti-NME7 AB Animals treated with antibodies did not show metastasis. Figures 46I-46P show photographs of the liver and lungs taken from animals after slaughter, as well as IVIS images. Figures 46I, 46K, 46M, and 46O are normal photographs. Figures 46J, 46L, 46N, and 46P are IVIS images revealing cancer cells that have metastasized thereto. As can be seen from the figures, anti-NME7 ABThe antibody significantly suppressed metastasis to the liver. The liver is a primary site for breast cancer metastasis. Figure 46Q is a bar graph of the number of photons emitted and counted by the IVIS detector for livers taken from control animals and treated animals. As can be seen from the inset graph of the IVIS measurements, the suppression of metastasis to the liver followed the order of metastasis when cells were injected into the tail vein, which is NME7 AB -MUC1 * This also coincides with the order of effectiveness in disrupting the interaction.

[0152] Figures 52A–52E show photographs of female nu / nu mice injected into the tail vein on day 0 with 10,000 luciferase-positive T47D metastatic breast cancer stem cells mixed with NME7AB to a final concentration of 32 nM. On days 1 and 2, the animals were injected with an additional 32 nM of NME7AB into their tail veins. AB Injection of 8F9A4A3, also known as 4A3, was administered intravenously to animals at a dose of 15 mg / kg. Here, the antibody was either the mouse sequence, the humanized 4A3 variant 4.8, or the humanized 4A3 variant 12.3. Figure 52A shows control animals injected with phosphate-buffered saline. Figure 52B shows anti-NME7 AB This shows animals treated with humanized 4A3 4.8. Figure 52C shows anti-NME7 AB This shows animals treated with humanized 4A3 12.3. Figure 52D shows anti-NME7 AB The images show animals treated with the monoclonal antibody mouse 4A3. Figure 52E shows a graph of the quantification of bioluminescence measurements of tumor volume obtained using the IVIS instrument. Animals were injected with either PBS or anti-NME7 antibody at a dose of 15 mg / kg every three days. Antibody hu4A34.8 indicates that sequences derived from human heavy chain antibody sequence 1.46 (SEQ ID NO: 1102) and light chain sequence 1.6 (SEQ ID NO: 1104) were used. Antibody hu4A312.3 indicates that sequences derived from human heavy chain antibody sequence 4.4 (SEQ ID NO: 1106) and light chain sequence 4.1 (SEQ ID NO: 1108) were used. Note that half of the animals treated with hu4A3 4.8 were given a partial half dose.

[0153] Humanized 4A3 variant 4.8 is human heavy chain IGHV1-46 * 01 (IMGT accession number X92343) and human light chain IGKV1-6 * It was generated from 01 (IMGT accession number M64858). Humanized 4A3 variant 4.8 was also called by 4A3146-16.

[0154] Humanized 4A3 variant 12.3 is human heavy chain IGHV4-4 * 01 (IMGT accession number X05713) and human light chain IGKV4-1 * It was generated from 01 (IMGT accession number Z00023). Humanized 4A3 variant 12.3 was also the caller 4A344-41.

[0155] The accession number is taken from the IMGT database (International ImMunoGeneTics Information System).

[0156] IGHVx-x or IGKVx-x correspond to the IMGT allele names. * x corresponds to the IMGT allele name.

[0157] The inventors performed immunofluorescence imaging on many cancer cell lines and found that cultured cancer cell lines were NME7 AB We determined whether or not it expresses MUC1. As clearly shown in Figures 47A-47F and 48A-48I, each MUC1-positive cancer cell line tested by the inventors was positive for NME7AB, and its binding was membranous, consistent with NME7AB secreted from cancer cells, and as a result it was found to express MUC1. *It binds to the extracellular domain. Figures 47A-47F show photographs of immunofluorescence experiments in which various cancer cell lines are stained for the presence of NME7AB. Figure 47A shows T47D breast cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47B shows ZR-75-1 breast cancer cells (also known as 1500s) stained with various concentrations of anti-NME7AB antibody 5D4. Figure 47C shows H1975 non-small cell lung cancer cells stained with various concentrations of anti-NME7 AB Figure 47E shows H292 non-small cell lung cancer cells stained with antibody 5D4. AB Figure 47F shows HPAFII pancreatic cancer cells stained with antibody 5D4. Figure 47F shows DU145 prostate cancer cells stained with various concentrations of anti-NME7AB antibody 5D4. As can be seen from the figures, all cancer cell lines tested by the inventors showed strong membrane staining against NME7AB. The monoclonal antibody used in these experiments was 5D4. In parallel, the same cell lines were stained using NME7AB antibodies 5A1 and 4A3, and the same results were obtained.

[0158] Figures 48A-48I show various human lung cancer cell lines in NME7 AB Figures 48A-48C show photographs of immunofluorescence experiments staining for the presence of [the substance]. AB Figure 48A shows H1975 non-small cell lung cancer cells, an adenocarcinoma, stained with antibody 5D4. Figure 48A shows DAPI and anti-NME7 staining. AB This is a staining overlay. Figure 48B shows anti-NME7 AB Single-color staining is shown. Figure 48C shows DAPI and anti-NME7 AB This is a magnified view of the staining overlay. Figures 48D-48F show various concentrations of anti-NME7. AB Figure 48D shows H292 non-small cell lung cancer cells, which are mucoepidermoid carcinomas, stained with antibody 5D4. AB This is a staining overlay. Figure 48E shows anti-NME7 AB The staining is shown individually. Figure 48F shows DAPI and anti-NME7 AB This is a magnified view of the staining overlay. Figures 48G-48I show various concentrations of anti-NME7.AB Figure 48G shows H358 non-small cell lung cancer cells, which are metastatic bronchoalveolar carcinoma, stained with antibody 5D4. AB This is a staining overlay. Figure 48H shows anti-NME7 AB The staining is shown individually. Figure 48I shows DAPI and anti-NME7 AB This is a magnified view of the staining overlay.

[0159] In addition, NME7 AB Culture of these cell lines in serum-free medium further enhanced the expression of their stem cells and metastasis markers. In particular, non-adherent cells, referred to here as suspensions, exhibited even higher expression of stem cells and metastasis markers than their adherent counterparts. Figures 49A-49I show NME7. AB The PCR graphs of cancer cell lines before and after culture are shown below: breast T47D, lung H1975, lung H358, and pancreatic HPAFII. Figure 49A shows the measured breast cancer metastasis marker CXCR4. Figure 49B shows the measured stem cell marker OCT4. Figure 49C shows the measured metastasis marker ALDH1. Figure 49D shows the measured stem cell marker SOX2. Figure 49E shows the measured stem cell marker NANOG. Figure 49F shows the measured metastasis marker CDH1 (also known as E-cadherin). Figure 49G shows the measured metastasis marker CD133. Figure 49H shows the measured stem cell marker ZEB2. Figure 49I shows the measured stem, cancer, and metastasis marker MUC1. Suspension cells (also known as tumor spheres) become able to independently proliferate scaffolds and show increased metastasis markers compared to adherent cells. Animals injected with cancer stem cells are NME7 AB These are animals injected with proliferating suspension cells. As can be seen from the figure, the metastasis marker, stem cell marker, or epithelial-mesenchymal transition (EMT) marker is NME7 AB The cells increased in number after culturing in the medium, indicating a transition to a more metastatic state. Figure 50 shows NME7 ABThe image shows an IVIS photograph of NSG mice at day 6, after 10-12 days in a culture containing NME7, in which 10,000 H358 lung cancer parent cells or H358 cells were injected into the tail vein. As can be seen from the figure, NME7 AB NCI-H358 lung cancer cells that proliferated within the NCI-H358 NME7 significantly increased their metastatic potential compared to the parent cells, which have been reported to be metastatic cells themselves. AB The functional increase in metastasis from metastatic cancer stem cells at 6 days is consistent with Figure 49, and H358 is NME7 AB This study demonstrates a significant increase in the expression of transfer markers after culturing in the medium.

[0160] Figure 51 shows the dimer NM23-H1 (also known as NME1), or NME7. AB The graphs show PCR results for the MUC1-negative prostate cancer strain PC3 before and after 2 or 3 passages in culture. The graphs show the difference in multiplicity levels of stem cells, cancer cell markers, and metastasis markers. As can be seen from the figure, NME1 or NME7 AB Repeated cultures within the cell line induce upregulation of stem, cancer, and metastasis markers, as well as a 5- to 8-fold upregulation of MUC1 expression.

[0161] In summary, these data indicate that NME7 lacking the DM10 domain is selected by cancer cells and binds to the extracellular domain of MUC1 lacking the tandem repeat domain, resulting in NME7 binding to MUC1 * We demonstrated that dimerization of the extracellular domain leads to increased cancer cell proliferation and enhanced metastatic potential. (NME7) AB and MUC1 * It is reasonable to assume that antibodies that disrupt the interaction between extracellular domains can suppress cancer cell proliferation and cancer metastasis. Here, the inventors of NME7 AB and MUC1 * Anti-NME7 inhibits interactions between extracellular domains. AB The antibody demonstrated that it actually suppresses cancer cell proliferation and cancer metastasis. Therefore, it is anti-NME7. ABAntibodies 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.

[0162] 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, making it suitable for use as an anti-NME7 for therapeutic purposes. AB The antibody is NME7 AB Alternatively, it is important that the antibody binds to NME7-X1 but not to NME1. In one aspect of the present invention, the antibody that appears to be optimal for therapeutic use is NME7 AB Alternatively, they were selected for their ability to bind to peptides specific to NME7-X1 and not present in the NME1 sequence. Figures 6-9 show NME7 AB List the peptides specific to this.

[0163] In a preferred embodiment, the antibody suitable for administration to a patient for the treatment or prevention of cancer or cancer metastasis is selected from the group of antibodies that bind to the NME7 B3 peptide. In a further preferred embodiment, the antibody suitable for administration to a patient for the treatment or prevention of cancer or cancer metastasis is selected from the group of antibodies that bind to the NME7 B3 peptide, NME7 AB The antibodies are selected from a group of antibodies that bind to NME7 but not to NME1. Examples of antibodies suitable for therapeutic use for the treatment or prevention of cancer or cancer metastasis, having demonstrated such anticancer and anti-metastatic activity in vitro and in vivo, include anti-NME7 antibodies 5A1, 4A3, and 5D4. These are merely examples, and antibodies produced as described herein and antibodies selected as described herein will have the same anticancer and anti-metastatic activity. Such antibodies may be complete antibodies or fragments thereof, including scFvs or antibody mimics, in which the variable domain of the antibody is incorporated into a protein scaffold that mimics the antibody. The antibodies may be from mouse, camel, llama, human or humanized human or non-human species, and may be monoclonal, polyclonal, scFvs or fragments thereof.

[0164] 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 or fragments thereof described herein can be administered to a patient as a standalone antibody or antibody fragment, or conjugated to a toxin such as an antibody-drug conjugate (ADC), or incorporated into a bispecific antibody or into a BiTE (bispecific T cell engager), or incorporated into a chimeric antigen receptor (CAR), or engineered to be expressed by cells that also express CARs. The cells may be immune cells, T cells, NK cells, or stem cells or progenitor cells, which can then be differentiated into T cells or NK cells.

[0165] Any antibody or fragment thereof described herein is thought to be an indicator of cancer or susceptibility to cancer, NME7 AB Alternatively, it can be used as a diagnostic agent to detect the presence of NME7-X1 in bodily fluids, cells, tissues, or body specimens. The diagnostic antibody may be attached to an imaging agent or nucleic acid tag and may be from any species, including camels, and may be used systemically or in bodily fluids such as blood, cells, or tissues, or in vitro, in vivo, or intraoperatively.

[0166] The selection criteria for therapeutically or diagnostically useful anti-NME7 antibodies depend on the format or form of treatment or diagnosis in which the antibody is incorporated. When the antibody or antibody fragment is administered to a patient as a standalone agent for the treatment or prevention of cancer or cancer metastasis, the antibody is i) NME7 AB ii) Binds to NME7-X1 but not to NME1; ii) Binds to PSMGFR peptide; iii) Binds to N-10 peptide; and iv) Binds to NME7 AB Alternatively, NME7-X1 and MUC1 * Interactions between extracellular domains, or NME7 ABAlternatively, the antibody may be selected for its ability to disrupt the interaction between NME7-X1 and N-10. The antibody may also be selected for its ability to bind to the NME7 B3 peptide. This therapeutic format also includes cells that have been engineered to express CAR and the selected anti-NME7 antibody.

[0167] Other forms require different selection criteria for anti-NME7 antibodies. If the anti-NME7 antibody is incorporated into an ADC, the ADC must be internally translocated by the target cells to induce target cell death. AB Alternatively, NME7-X1 is MUC1 * It should be noted that the antibody binds to the extracellular domain of MUC1. * If the binding of NME to the extracellular domain is disrupted, the toxin-complexed antibody will be internalized and the cells will not be killed. Similarly, if an anti-NME7 antibody is incorporated into CAR or BiTE, NME7 AB Alternatively, the interaction between NME7 and X1 cannot be interrupted, or immune cells may no longer be able to direct the killing agent towards cancer cells. When anti-NME7 antibodies are used as diagnostic agents, NME7 AB Alternatively, the interaction between NME7 and X1 cannot be interrupted, or the antibody and associated label will be washed away. Therefore, in the case of ADC, CAR T, or CAR-NK, BiTE, or diagnostic applications, the anti-NME7 antibody is i) NME7 AB ii) Binds to NME7-X1 but not to NME1; ii) Binds to PSMGFR peptide; iii) Binds to N-10 peptide; and iv) Binds to MUC1 * Interaction with the extracellular domain, or NME7 AB Alternatively, without disrupting the interaction between NME7-X1 and the N-10 peptide, NME7 AB Alternatively, the antibody may be selected for its ability to bind to NME7-X1. The antibody may also be selected for its ability to bind to the NME7 B3 peptide.

[0168] In one aspect of the present invention, cells are anti-NME7 of the present invention. ABThe cells are manipulated to express an antibody or a fragment thereof. The cells may be immune cells such as T cells or NK cells, or stem cells or progenitor cells, which can differentiate into more mature immune cells such as T cells or NK cells. In a preferred embodiment, anti-NME7 AB Cells manipulated to express antibodies are also manipulated to express chimeric antigen receptors (CARs). In a preferred embodiment, the CAR recognizes tumor-associated antigens. In a preferred embodiment, the CAR is MUC1 * It targets. In a more preferred embodiment, CAR is anti-MUC1 * The antibody MNC2 is directed towards the tumor. In another aspect of the present invention, cells engineered to express CAR are also engineered to inducibly express an anti-NME7 antibody. In one example, anti-NME7 AB The nucleic acid encoding the antibody is inserted into the Foxp3 enhancer or promoter. Another example is anti-NME7. AB The antibody is present in the NFAT induction system. In one embodiment, the NFAT induction system is anti-NME7 AB The NFATc1 response element is inserted upstream of the antibody sequence. These can be inserted into the IL2 promoter, Foxp3 enhancer or promoter, or other suitable promoter or enhancer.

[0169] In another aspect of the present invention, NME7 AB Alternatively, a peptide specific to NME7-X1 may be 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 the NME7 B3 peptide having the Cys-14-Ser mutation.

[0170] Another aspect of the present invention is the anti-NME7 in host animals. ABThe method includes a method for generating antibodies, in which animals are immunized with the NME7 B3 peptide. In a preferred embodiment, the NME7 B3 peptide has a serine-mutated cysteine ​​14 (SEQ ID NO: 169) to avoid the formation of a disulfide bond that suppresses NME7-specific antibody production.

[0171] Another aspect of the present invention involves cells in NME7 AB The method includes generating cells with enhanced metastatic potential, which may involve culturing them with NME7-X1. These cells can then be used in many aspects of drug discovery.

[0172] Another aspect of the present invention is anti-NME7 AB This also includes cells that are engineered to express NME7-X1. AB Alternatively, NME7-X1 is a human sequence. Their expression may be inducible. In one embodiment, the cell is an egg, which is human NME7 AB Alternatively, it may develop into an animal that can express NME7-X1.

[0173] NME7 is MUC1 * It binds to the extracellular domain of the growth factor receptor and dimerizes it. Tissue studies are conducted using MUC1. * However, it is shown that it increases with increasing tumor malignancy and metastasis.Here, the inventors show that NME7 expression increases with increasing tumor malignancy and metastasis (Figures 39-41).Here, the inventors show that NME7 and MUC1 * Antibodies that inhibit the interaction between these molecules have been shown to suppress tumor growth and metastasis.

[0174] Other NME family members are MUC1 * It can bind to the extracellular domain of growth factor receptors and dimerize them. For example, the inventors have shown that NME1, NME2, and NME6 exist as dimers, and that they are MUC1 * We demonstrated that it binds to extracellular domains and dimerizes them. NME7 AB And NME7-X1 is MUC1 *It has two domains that can bind to the extracellular domain, and therefore they are used as monomers for MUC1 * The growth factor receptor is dimerized and activated. The inventors hereby demonstrate that an anti-NME7 antibody suppresses cancer and cancer metastasis. Similarly, antibodies or antibody mimetics that bind to these other NME proteins may be anticancer or anti-metastatic therapeutic agents that can be administered to patients diagnosed with cancer or metastasis, or patients at risk of developing cancer or metastasis. In one aspect of the present invention, an antibody that can be used therapeutically for the treatment of cancer or metastasis is an antibody that binds to NME1, NME2, NME3, NME4, NME5, NME6, NME7, NME8, NME9, or NME10. In one aspect of the present invention, a therapeutic antibody or antibody mimetics inhibits the binding of NME proteins and their homologous growth factor receptors. In one aspect of the present invention, a therapeutic antibody or antibody mimetics binds NME proteins and MUC1 * It suppresses the interaction of the extracellular domains. In another aspect of the present invention, the therapeutic antibody or antibody mimetic binds to a peptide derived from NME1, NME2, NME3, NME4, NME5, NME6, NME7, NME8, NME9, or NME10, the peptide being homologous to the NME7 A1, A2, B1, B2, or B3 peptide.

[0175] The following is a sequence alignment showing homology and identity alignment between NME7 and other NME family members. Underlined or underlined and bolded 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).

[0176] Nucleoside diphosphate kinase 7 isoform a [Homo sapiens] (Hu_7) [ka] >NME2 Theoretical pI / Mw:8.52 / 17298.04 [ka] Global / Global (NW) score: 171; 155aa overlap (1-131:1-152), 26.5% identical (56.8% similar). [ka] Global / Global (NW) score: 104; 156aa overlap (1-134:1-152), 24.4% identical (51.3% similar). [ka] >NME3 Theoretical pI / Mw:5.96 / 19088.97 [ka] >NME4 Theoretical pI / Mw:10.30 / 20658.59 [ka] 133aa overlap (1~131:56~185): 29.3% identical (68.4% similar) [ka] 132aa overlap (3~134:40~167): 28.8% identical (56.8% similar) [ka] >NME5 Theoretical pI / Mw:6.08 / 29296.23 [ka] 131aa overlap (1~131:13~143): 44.3% identical (74.8% similar) [ka] 132aa overlap (3~134:15~143): 28.0% identical (58.3% similar) [ka] >NME6 Theoretical pI / Mw:7.81 / 22003.16 [ka] 133aa overlap (3~131:22~153): 37.6% identical (68.4% similar) [ka] 133aa overlap (3~134:22~153): 29.3% identical (57.9% similar) [ka] >NME8 Theoretical pI / Mw:4.90 / 67269.94 [ka] 133aa overlap (1~131:316~448): 36.1% identical (69.2% similar) [ka] Waterman-Eggert score: 269; 85.9 bits; E(1) < 1.1e-21 128aa overlap (1~127:451~577): 33.6% identical (72.7% similar) [ka] Waterman-Eggert score: 119; 40.4 bits; E(1) < 5.3e-08 65aa overlap (3~65:156~220): 33.8% identical (73.8% similar) [ka] 116aa overlap (3~118:453~566): 33.6% identical (65.5% similar) [ka] Waterman-Eggert score: 128; 41.3 bits; E(1) < 2.9e-08 116aa overlap (20~134:334~448): 22.3% identical (60.3% similar) [ka] >--Waterman-Eggert score: 76; 26.4 bits; (E(1)<0.00088 111aa overlap (6~105:159~268): 23.4% identical (46.8% similar) [ka] >NME9 [ka] 46aa overlap (3~46:100~145): 41.3% identical (67.4% similar) [ka] >--Waterman-Eggert score: 30; 13.5 bits; (E(1)<0.85 14aa overlap (69~82:100~113): 28.6% identical (71.4% similar) [ka] >--Waterman-Eggert score: 29; 13.2 bits; (E(1)<0.91 31aa overlap (12~42:121~149): 25.8% identical (74.2% similar) [ka] 53aa overlap (1-53:98-150): 39.6% identical (69.8% similar) [ka] >NME10 NP_008846.2 Protein XRP2 [Homo sapiens] [ka] 68aa overlap (11~78:246~308): 23.5% identical (66.2% similar) [ka] >Waterman-Eggert score: 35; 15.1 bits; (E(1)<0.73 45aa overlap (66~108:200~244): 28.9% identical (57.8% similar) [ka] >--Waterman-Eggert score: 33; 14.4 bits; (E(1)<0.87 75aa overlap (7~80:35~109) - 14.7% identical (52.0% similar) [ka] >--Waterman-Eggert score: 45; 17.5 bits; (E(1)<0.22 51aa overlap (4~50:130~180): 21.6% identical (58.8% similar) [ka]

[0177] For example, antibodies or antibody mimes that bind to peptides homologous to NME7 ("homologous peptides"), particularly peptides homologous to A1, A2, B1, B2, or B3 peptides, may be administered to patients diagnosed with cancer or cancer metastasis, or patients at risk of developing cancer or cancer metastasis.

[0178] Peptides homologous to peptides A1, A2, B1, B2, or B3 Peptides homologous to peptides A1, A2, B1, B2, or B3 may include, but are not limited to, the following:

[0179] NME2A1 (amino acid) RASEEHLKQHYIDLKD (Sequence ID 247)

[0180] NME2A2 (amino acid) PADSKPGT (Sequence ID 248)

[0181] NME2B1 (amino acid) QKGFRLVAMKFLRASEEHLK (Sequence ID 249)

[0182] NME2B2 (amino acid) IDLKDRPFPGLVKY (Sequence ID 250)

[0183] NME2B3 (amino acid) GDFCIQVGRNIIHGSDSVKSAEKEISLWF (Sequence ID 251)

[0184] NME3A1 (amino acid) QASEELLREHYVELRE (Sequence ID 252)

[0185] NME3A1 (amino acid) PGDATPGT (SEQ ID NO: 253)

[0186] NME3B1 (amino acid) RKGFKLVALKLVQASEELLR (Sequence ID 254)

[0187] NME3B2 (amino acid) VELRERPFYSRLVKY (Sequence ID 255)

[0188] NME3B3 (amino acid) GDFCVEVGKNVIHGSDSVESAQREIALWF (Sequence ID 256)

[0189] NME4A1 (amino acid) QAPESVLAEHYQDLRR (Sequence ID 257)

[0190] NME4A2 (amino acid) SAEAAPGT (Sequence ID 258)

[0191] (amino acid) RRGFTLVGMKMLQAPESVLA (Sequence ID 259)

[0192] NME4B2 (amino acid) QDLRRKPFYPALIRY (Sequence ID 260)

[0193] NME4B3 (amino acid) GDFSVHISRNVIHASDSVEGAQREIQLWF (Sequence ID 261)

[0194] NME5A1 (amino acid) RLSPEQCSNFYVEKYG (Sequence ID 262)

[0195] NME5A2 (amino acid) SLVAKETHPDS (Sequence ID 263)

[0196] NME5B1 (amino acid) RSGFTIVQRRKLRLSPEQCS (Sequence ID 264)

[0197] NME5B2 (amino acid) VEKYGKMFFPNLTAY (Sequence ID 265)

[0198] NME5B3 (amino acid) AIYGTDDLRNALHGSNDFAAAEREIRFMF(Sequence ID 266)

[0199] NME6A1 (amino acid) LWRKEDCQRFYREHEG (Sequence ID 267)

[0200] NME6A2 (amino acid) VFRARHVAPDS (Sequence ID 268)

[0201] NME6B1 (amino acid) SNKFLIVRMRELLWRKEDCQ (Sequence ID 269)

[0202] NME6B2 (amino acid) REHEGRFFYQRLVEF (Sequence ID 270)

[0203] NME6B3 (amino acid) GSFGLTDTRNTTHGSDSVVSASREIAAFF (Sequence ID 271)

[0204] NME8A1 (amino acid) VLSEKEAQALCKEYEN (Sequence ID 272)

[0205] NME8A2 (amino acid) VEEAIEYFPES (Sequence ID 273)

[0206] NME8A3 (amino acid) FLTPEQIEKIYPKVTG (Sequence ID 274)

[0207] NME8A4 (amino acid) PEEAKLLSPDS (Sequence ID 275)

[0208] NME8A5 (amino acid) VLTEEQVVNFYSRIAD (Sequence ID 276)

[0209] NME8B1 (amino acid) EAGFDLTQVKKMFLTPEQIE (Sequence ID 277)

[0210] NME8B2 (amino acid) PKVTGKDFYKDLLEM (Sequence ID 278)

[0211] NME8B3 (amino acid) AQFGISKLKNIVH (Sequence ID 279)

[0212] NME8B4 (amino acid) DEDFKILEQRQVVLSEKEAQ (Sequence ID 280)

[0213] NME8B5 (amino acid) KEYENEDYFNKLIEN (Sequence ID 281)

[0214] NME8B6 (amino acid) AQFAMDSLPVNQLYGSDSLETAEREIQHFF (Sequence ID 282)

[0215] NME8B7 (amino acid) KAGFIIEAEHKTVLTEEQVV (Sequence ID 283)

[0216] NME8B8 (amino acid) SRIADQCDFEEFVSF (Sequence ID 284)

[0217] NME9A1 (amino acid) TMTEAEVRLFY (Sequence ID 285)

[0218] NME9B1 (amino acid) EAGFEILTNEERTMTEAEVR (Sequence ID 286)

[0219] NME10A1 (amino acid) SMKAEDAQRVFREK (Sequence ID 287)

[0220] NME10A2 (amino acid) GQRQKSSDES (Sequence ID 288)

[0221] NME10A3 (amino acid) IQDCENCNIYIFDHSA (Sequence ID 289)

[0222] NME10B1 ELAFQFKDAGLSIFNNTWSNIH(Sequence ID 290)

[0223] In some cases, peptides derived from other NME proteins can be homologous to NME7 A1, A2, B1, B2, or B3 peptides by shifting the frame or by elongating the NME7 peptide so that the elongated peptide is homologous to the NME7 peptide that produces antibodies that suppress cancer or cancer metastasis. As another example, antibodies or antibody mimes that bind to an elongated peptide homologous to NME7 ("elongated peptide") may be administered to patients diagnosed with cancer or cancer metastasis, or patients at risk of developing cancer or cancer metastasis.

[0224] Elongated peptide homologous to peptides A1, A2, B1, B2, or B3 Peptides homologous to the elongated peptides A1, A2, B1, B2, or B3 may include, but are not limited to, the following:

[0225] NME2A1 (amino acid) RASEEHLKQHYIDLKDRPFFPGL (Sequence ID 291)

[0226] NME2A2 (amino acid) LGETNPADSKPGTIRGDF (Sequence ID 292)

[0227] NME2B1 (amino acid) GLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHY (Sequence ID 293)

[0228] NME2B2 (amino acid) YIDLKDRPFFPGLVKYMNSGPVVAM (Sequence ID 294)

[0229] NME2B3 (amino acid) PGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWF (Sequence ID 295)

[0230] NME3A1 (amino acid) LKLVQASEELLREHYVELRERPFYSRL (Sequence ID 296)

[0231] NME3A1 (amino acid) LIGATDPGDATPGTIRGDF (Sequence ID 297)

[0232] NME3B1 (amino acid) LVGEIVRRFERKGFKLVALKLVQASEELLRE (Sequence ID 298)

[0233] NME3B2 (amino acid) EHY-VELRERPFYSRLVKYMGSGPVVAM (Sequence ID 299)

[0234] (amino acid) PGTIRGDFCVEVGKNVIHGSDSVESAQREIALWF (Sequence ID 300)

[0235] NME4A1 (amino acid) GFTLVGMKMLQAPESVLAEHYQDLRRKPF (Sequence ID 301)

[0236] NME4A2 (amino acid) GHTDSAEAAPGTIRGDF (Sequence ID 302)

[0237] NME4B1 (amino acid) LVGDVIQRFERRGFTLVGMKMLQAPESVLAEHY (Sequence ID 303)

[0238] NME4B2 (amino acid) EHYQDLRRKPFYPALIRYMSSGPVVAM (Sequence ID 304)

[0239] NME4B3 (amino acid) PGTIRGDFSVHISRNVIHASDSVEGAQREIQLWF (Sequence ID 305)

[0240] NME5A1 (amino acid) GFTIVQRRKLRLSPEQCSNFYVEKYGKMFF (Sequence ID 306)

[0241] NME5A2 (amino acid) LLGPNNSLVAKETHPDSLRAIYGTD (Sequence ID 307)

[0242] NME5B1 (amino acid) IQDIILRSGFTIVQRRKLRLSPEQCSNFY (Sequence ID 308)

[0243] NME5B2 (amino acid) FYVEKYGKMFFPNLTAYMSSGPLVAM (Sequence ID 309)

[0244] NME5B3 (amino acid) PDSLRAIYGTDDLRNALHGSNDFAAAEREIRFMF(Sequence ID 310)

[0245] NME6A1 (amino acid) FLIVRMRELLWRKEDCQRFYREHEGRFFYQRL (Sequence ID 311)

[0246] NME6A2 (amino acid) LMGPTRVFRARHVAPDSIRGSFG (Sequence ID 312)

[0247] NME6B1 (amino acid) ILSNKFLIVRMRELLWRKEDCQRFY (Sequence ID 313)

[0248] NME6B2 (amino acid) FYREHEGRFFYQRLVEFMASGPIRA (Sequence ID 314)

[0249] NME6B3 (amino acid) ARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFF (Sequence ID 315)

[0250] NME8A1 (amino acid) FKILEQRQVVLSEKEAQALCKEYENEDYFNKLI (Sequence ID 316)

[0251] NME8A2 (amino acid) WKQLLGPRTVEEAIEYFPESLCAQFAMD (Sequence ID 317)

[0252] NME8A3 (amino acid) AGFDLTQVKKMFLTPEQIEKIYPKVTGKDFYKDL (Sequence ID 318)

[0253] NME8A4 (amino acid) EWRRLMGPTDPEEAKLLSPDSIRAQFG (Sequence ID 319)

[0254] NME8A5 (amino acid) KAGFIIEAEHKTVLTEEQVVNFYSRIADQCDFEE(Sequence ID 320)

[0255] NME8B1 (amino acid) ILKIVKEAGFDLTQVKKMFLTPEQIEKIY (Sequence ID 321)

[0256] NME8B2 (amino acid) YPKVTGKDFYKDLLEMLSVGP(Sequence ID 322)

[0257] NME8B3 (amino acid) DPEEAKLLSPDSIRAQFGISKLKNIVH(Sequence ID 323)

[0258] NME8B4 (amino acid) LRIIKDEDFKILEQRQVVLSEKEAQ (Sequence ID 324)

[0259] NME8B5 (amino acid) KEYENEDYFNKLIENMTSGPSLA (Sequence ID 325)

[0260] (amino acid) PESLCAQFAMDSLPVNQLYGSDSLETAEREIQHFF (Sequence ID 326)

[0261] NME8B7 (amino acid) IKRKITKAGFIIEAEHKTVLTEEQVVNFY(Sequence ID 327)

[0262] NME8B8 (amino acid) FYSRIADQCDFEEFVSFMTSG (Sequence ID 328)

[0263] NME9A1 (amino acid) AGFEILTNEERTMTEAEVRLFY (Sequence ID 329)

[0264] NME9B1 (amino acid) IIMKIQEAGFEILTNEERTMTEAEVRLFY (Sequence ID 330)

[0265] NME10A1 (amino acid) GFFLVQTKEVSMKAEDAQRVFREKAP (Sequence ID 331)

[0266] NME10A2 (amino acid) EANRSIVPISRGQRQKSSDESCLVVLFAGD(Sequence ID 332)

[0267] NME10A3 (amino acid) IQDCENCNIYIFDHSA (Sequence ID 333)

[0268] NME10B1 ELAFQFKDAGLSIFNNTWSNIHDFTPVDCT(Sequence ID 334)

[0269] Some NME proteins perform functions necessary for normal cell proliferation 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 targeted cancer-associated NMEs but not to untargeted NMEs. For example, the anti-NME7 antibodies presented here, 8F9A5A1, 8F9A4A3, and 5F3A5D4, are NME7 antibodies. AB They are selected for their ability to bind to but not to NME1; and they are further selected based on their ability to suppress cancer and cancer metastasis.

[0270] In another aspect of the present invention, an anti-NME7 antibody, an antibody fragment, e.g., scFv, or an antibody mimetic fragment is incorporated into a chimeric antigen receptor (CAR) that is engineered to be expressed in immune cells. The immune cells then use an anti-NME7 CAR and an anti-MUC1 * CARs, or both, can be manipulated to express. One of the CARs can be expressed from an inducible promoter. Alternatively, immune cells can express MUC1 * CARs and other CARs, as well as inducible anti-NME7 antibodies or antibody fragments, can be manipulated to express them. In some cases, the inducible promoter may include an NFAT response element. In one embodiment, these manipulated species are expressed in T cells, NK cells, or dendritic cells. Immune cells can be obtained from a patient or a donor. In some embodiments, immune molecules such as MHC, checkpoint inhibitors, or receptors for checkpoint inhibitors are mutagenesized or removed, for example, using CrisPR or CrisPR-like technologies. In another embodiment, ITAM molecules, Fos, or Jun are mutagenesized or genetically excised in patient or donor-derived immune cells using Talen, Sleeping Beauty, CrisPR, or CrisPR-like technologies.

[0271] In one aspect of the present invention, an anti-NME7 antibody or antibody mimetic for use in CAR T format is specific to NME7 but MUC1 * The antibody or antibody mimetic is selected from a group of antibodies or antibody mimetics that do not disrupt the binding of NME7 to the extracellular domain of NME7. Thus, an anti-NME7 antibody or antibody mimetic that directs CAR T cells to the tumor will not simply deprive the ligand of the receptor, but will also prevent the T cells from injecting granzyme B into the target cancer cells. Such antibodies or antibody mimetics are produced by immunizing animals with NME7 peptides such as NME7 peptides A1, A2, B1, B2, or B3, or are selected for their ability to bind to NME7 peptides A1, A2, B1, B2, or B3. The antibody or antibody mimetic is selected for their ability to specifically bind to NME7 but not to NME1 or NME2, and for their ability to bind to NME7 and MUC1 * The binding between extracellular domains can be screened for their inability to be cleaved. For example, in an ELISA instrument, the PSMGFR peptide is immobilized on the surface. Labeled NME7 AB This is surface-immobilized MUC1 * It is bound to the extracellular domain, NME7 AB The detection of the label is measured in the presence or absence of the test antibody or antibody mimetic. In one aspect of the present invention, NME7 AB and surface-immobilized MUC1 * Antibodies that do not reduce binding to extracellular domain peptides are selected as antibodies to be incorporated into CARs, engineered to be expressed in immune cells, and then administered to patients for the treatment or prevention of cancer or cancer metastasis.

[0272] In one aspect of the present invention, an anti-NME7 antibody or a 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 A1, A2, B1, B2, or B3 peptides” and “Extended peptides homologous to A1, A2, B1, B2, or B3 peptides”.

[0273] In another embodiment, 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 embodiment, the antibody, antibody fragment, or antibody mimetic binds to an NME7 peptide containing most or all of the B3 peptide. In one embodiment of the present invention, the anti-NME7 antibody, antibody fragment, or antibody mimetic contains a sequence derived from the variable domain of the anti-NME7 antibodies 8F9A4A3 (SEQ ID NOs: 1001-1015), 8F9A5A1 (SEQ ID NOs: 1016-1030), or 8H5H5G4 (SEQ ID NOs: 1031-1045), as shown below.

[0274] Anti-NME7 B3 peptide monoclonal antibody Monoclonal antibody 8F9A4A3 "4A3" Heavy chain variable region sequence Gaggtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccaga agttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 386)

[0275] Translational proteins where the underlined sequence is the complementarity-determining region (CDR). EVQLQQSGPELVKPGASVKISCKTSG NTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVSS (Sequence ID 387)

[0276] Mouse 8F9A4A3 heavy chain variable domain framework 1 (FR1) sequence gaggtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctgga (Sequence number) EVQLQQSGPELVKPGASVKISCKTSG(sequence ID)

[0277] Heavy chain variable region CDR1: aacacattcactgaatacaccatgcac NTFTEYTMH (Sequence ID 388)

[0278] Mouse 8F9A4A3 heavy chain variable domain framework 2 (FR2) sequence tgggtgaagcagagccatggaaagagccttgagtggattgga(sequence number) WVKQSHGKSLEWIG(sequence number )

[0279] Mouse 8F9A4A3 heavy chain variable region CDR2: ggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggc(sequence array ) GFNPNNGVTNYNQKFKG (Sequence ID 389)

[0280] Mouse 4A3 heavy chain variable domain framework 3 (FR3) sequence aaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaaga (SEQ ID NO.) KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR(Sequence ID)

[0281] Mouse 8F9A4A3 heavy chain variable region CDR3: cggtactaccatagtctctacgtgttttactttgactac (Syntax ) RYYHSLYVFYFDY (Sequence ID 390)

[0282] Mouse 8F9A4A3 light chain variable region photo gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagatggaactgttgaactcctgatcttttacacatcaagtttacac tcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggaaataaaa (SEQ ID NO: 391)

[0283] Translational proteins where the underlined sequence is the complementarity-determining region (CDR). DIQMTQTTSSLSASLGDRVTLSCSASQGISNYLNWYQQKPDGTVELLIFYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIK(Sequence ID 392)

[0284] Mouse 8F9A4A3 light chain variable domain framework 1 (FR1) sequence gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgc (Sequence number) DIQMTQTTSSLSASLGDRVTLSC(sequence ID)

[0285] Mouse 8F9A4A3 light chain variable region CDR1: agtgcaagtcagggcattagcaattatttaaac(sequence number) SASQGISNYLN (Sequence ID 393)

[0286] Mouse 4A3 light chain variable domain framework 2 (FR2) sequence tggtatcagcagaaaccagatggaactgttgaactcctgatcttt(sequence) WYQQKPDGTVELLIF(sequence number)

[0287] Mouse 8F9A4A3 light chain variable region CDR2: tacacatcaagtttacactca(sequence number) YTSSLHS (Sequence ID 394)

[0288] Mouse 8F9A4A3 light chain variable domain framework 3 (FR3) sequence ggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt (Sequence number) GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC(sequence number)

[0289] Mouse 8F9A4A3 light chain variable region CDR3: Cagcagtatagtaagcttccttacacg (sequence number) QQYSKLPYT (Sequence ID 395)

[0290] Humanized 8F9A4A3 H-ori heavy chain variable domain sequence caggttcagctggttcagtctggtgcagaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaacca gaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct(Sequence number ) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(Sequence ID )

[0291] Humanized 8F9A4A3 H-1.46 heavy chain variable domain sequence caagtgcagctggtgcagagcggcgccgaggtgaagaaacctggcgccagcgtgaaagtgtcctgcaaggccagcggcaatacattcaccgagtacacaatgcactgggtcagacaggcccccggccagggcctggaatggatcggcggatttaaccccaacaacggcgtgacaaactacaaccagaa gttcaagggcaaggtgaccatcacaagagacaccagcagcagcaccgtgtacatggaactgtcttctctgcggagcgaggataccgccgtgtactattgtgccagacggtactaccacagcctgtacgtgttctacttcgactactggggacagggcaccctggttaccgtgtcctct (SEQ ID NO: 1101) QVQLVQSGAEVKKPGASVKVSCKASGNTFTEYTMHWVRQAPGQGLEWIGGFNPNNGVTNYNQKFKGKVTITRDTSSSTVYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (Sequence ID 1102)

[0292] Humanized 8F9A4A3 H-3.15 heavy chain variable domain sequence gaggtgcagctggtggaaagcggcggcggcctggttaagcctggcggatctctgagactgagctgtgccgcttctggcaataccttcaccgagtacaccatgcactgggtgcggcaggcccctggaaaaggcctggaatggatcggcggatttaaccccaacaacggcgtgacaaattacaacca gaaattcaagggcaagttcaccatcacaagagataagagcaagaacaccctgtacctgcaaaatgaacagcctgaagtccgaggacaccgccgtgtactactgcgccagacggtactaccacagcctctatgtgttctacttcgactactggggccagggcacactggtcaccgtgtccagc(Sequence number ) EVQLVESGGGLVKPGGSLRLSCAASGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKFTITRDKSKNTLYLQMNSLKSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(Sequence ID)

[0293] Humanized 8F9A4A3 H-4.4 heavy-chain variable domain sequence caagtgcagctgcaggagagcggacctggcctggttaagcctggaggcaccctgtctctgacatgtgctgtgtctggcaatacctttaccgagtacaccatgcactgggtgcggcagcctccaggcaagggcctggaatggatcggcggcttcaaccccaacaacggcgtgacaaattacaaccagaaattcaagggaaaagtgaccatcaccgtggataagtccaagaacaccttcagcctcaagctgagcagcgtgacagccgccgacaccgccgtgtactactgcgccagaagatactatcacagcctgtacgtgttctacttcgactactggggccagggcacactggtcaccgtgtccagc (SEQ ID NO: 1105) QVQLQESGPGLVKPGGTLSLTCAVSGNTFTEYTMHWVRQPPGKGLEWIGGFNPNNGVTNYNQKFKGKVTITVDKSKNTFSLKLSSVTAADTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1106)

[0294] Humanized 8F9A4A3 L-1.6 heavy chain variable domain sequence gatatccagatgacacagagccctagctccctgagcgccagcgtgggcgaccgggtcaccattacatgcagcgcttctcagggcatctccaactacctgaactggtaccagcagaaacccggcaaggcccctaagctgctgatcttctacaccagctctctgcacagcggcgtgccatctagattcagcggatctggcagcggcaccgactacaccctgaccatcagctccctccagcctgaggacttcgccacctactactgtcagcaatacagcaagctgccttatacctttggcggcggaacaaaggtggaaatcaag (SEQ ID NO: 1103) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIFYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKVEIK(Sequence ID 1104)

[0295] Humanized 8F9A4A3 L-3.15 heavy chain variable domain sequence gagatcgtgatgacccagagcccagctacacttagtgtgagtccaggtgaacgggctaccctgtcctgcagcgccagccagggcatcagcaactacctgaactggtaccagcagaaacctggccaggcccctagactgctgatcttctacaccagcagcctgc acagcggcatccccgccagattcagcggcagcggctctggaacagactacaccctgacaatctctagcctgcagtctgaagattttgccgtctactactgtcagcaatacagcaagctgccttataccttcggcggcggaaccaaggtggaaattaag(SEQ ID NO. ) EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWYQQKPGQAPRLLIFYTSSLHSGIPARFSGSGSGTDYTLTISSLQSEDFAVYYCQQYSKLPYTFGGGTKVEIK(Sequence ID )

[0296] Humanized 8F9A4A3 L-4.1 light chain variable domain sequence gatatcgtgatgacccagagcccagacagcctggcagtgagtctgggtgagcgtgctacaatcaactgcagcgccagccagggcatctccaactacctgaattggtatcagcagaaacctggccaggctcctaagctgctgatcttctacaccagcagcctgcac agcggcgtgccagatagattcagcggcagcggatctggcaccgactacacactgaccatttcttctctccaggccgaggacgtggccgtctactactgtcagcaatacagcaagctgccttacacctttggcggaggcacaaaggtggaaatcaag (SEQ ID NO: 1107) DIVMTQSPDSLAVSLGERATINCSASQGISNYLNWYQQKPGQAPKLLIFYTSSLHSGVPDRFSGSGSGTDYTLTISSLQAEDVAVYYCQQYSKLPYTFGGGTKVEIK (Sequence ID 1108)

[0297] Monoclonal antibody 5F3A5D4 Heavy chain variable region sequence H-2,3,4,13,15 gtccagctgcaacagtctggacctgatctggtgaagcctggagacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaag ttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 427)

[0298] Translation proteins in which the underlined sequence is the complementarity-determining region (CDR): VQLQQSGPDLVKPGTSVKISCKTSG NTFTEYTMH WVKQSHGKSLEWIG GFNPNNGVTNYNQKFKG KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR RYYHSTYVFYFDS WGQGTTLTVSS (Sequence ID 428)

[0299] Heavy chain variable region CDR1: NTFTEYTMH (Sequence ID 429)

[0300] Heavy chain variable region CDR2: GFNPNNGVTNYNQKFKG (Sequence ID 430)

[0301] Heavy chain variable region CDR3: RYYHSTYVFYFDS (Sequence ID 431)

[0302] Light chain variable region arrangement K-1, 2, 3, 4, 9 gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacatt caggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 432)

[0303] Translation proteins in which the underlined sequence is the complementarity-determining region (CDR): DIQMTQTTSSLSASLGDRVTISC SASQGISNYLN WFQQKPDGTIKLLIY YTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYC QQYSKLPYT FGGGTKLEIKR (Sequence ID 433)

[0304] Light chain variable region CDR1: SASQGISNYLN (Sequence ID 434)

[0305] Light chain variable region CDR2: YTSSLHS (Sequence ID 435)

[0306] Light chain variable region CDR3: QQYSKLPYT (Sequence ID 436)

[0307] Mouse 5F3A5D4-V2 heavy-chain variable domain sequence: (DNA) gaggtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaac cagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (amino acid) EVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS

[0308] Mouse 5F3A5D4-V2 heavy-chain variable domain framework 1 (FR1) sequence (DNA) gaggtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctgga (amino acid) EVQLQQSGPDLVKPGTSVKISCKTSG

[0309] Mouse 5F3A5D4-V2 heavy chain variable domain complementarity determination region 1 (CDR1) sequence (DNA) aacacattcactgaatacaccatgcac (amino acid) NTFTEYTMH

[0310] Mouse 5F3A5D4-V2 heavy-chain variable domain framework 2 (FR2) sequence (DNA) tgggtgaagcagagccatggaaagagccttgagtggattgga (amino acid) WVKQSHGKSLEWIG

[0311] Mouse 5F3A5D4-V2 heavy chain variable domain complementarity determination region 2 (CDR2) sequence (DNA) ggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggc (amino acid) GFNPNNGVTNYNQKFKG

[0312] Mouse 5F3A5D4-V2 heavy-chain variable domain framework 3 (FR3) sequence (DNA) aaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaaga (amino acid) KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

[0313] Mouse 5F3A5D4-V2 heavy chain variable domain complementarity determination region 3 (CDR3) sequence (DNA) cgttactaccatagtacctacgtgttctactttgactcc (amino acid) RYYHSTYVFYFDS

[0314] Mouse 5F3A5D4-V2 heavy-chain variable domain framework 4 (FR4) sequence (DNA) tggggccaaggcaccactctcacagtctcctca (amino acid) WGQGTTLTVSS

[0315] Mouse 5F3A5D4-2 light chain variable domain sequence (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtt tacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaa (amino acid) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIK

[0316] Mouse 5F3A5D4-2 Light Chain Variable Domain Framework 1 (FR1) Sequence (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgc (amino acid) DIQMTQTTSSLSASLGDRVTISC

[0317] Mouse 5F3A5D4-2 light chain variable domain complementarity determination region 1 (CDR1) sequence (DNA) agtgcaagtcagggcattagcaattatttaaac (amino acid) SASQGISNYLN

[0318] Mouse 5F3A5D4-2 Light Chain Variable Domain Framework 2 (FR2) Sequence (DNA) tggtttcagcagaaaccagatggaactattaagctcctgatctat (amino acid) WFQQKPDGTIKLLIY

[0319] Mouse 5F3A5D4-2 light chain variable domain complementarity determination region 2 (CDR2) sequence (DNA) tacacatcaagtttacattca (amino acid) YTSSLHS

[0320] Mouse 5F3A5D4-2 Light Chain Variable Domain Framework 3 (FR3) Sequence (DNA) ggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgt (amino acid) GVPSRFSGSGSGTDYSLTISNVEPEDIATYYC

[0321] Mouse 5F3A5D4-2 light chain variable domain complementarity determination region 3 (CDR3) sequence (DNA) cagcagtatagtaagcttccttacacg (amino acid) QQYSKLPYT

[0322] Mouse 5F3A5D4-2 Light Chain Variable Domain Framework 4 (FR4) Sequence (DNA) ttcggaggggggaccaagctggagataaaa (amino acid) FGGGTKLEIK

[0323] Mouse 5F3A5D4-3 light chain variable domain sequence (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagatggaactgttgaactcctgatcttttacacatcaagtt tacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggaaataaaa (amino acid) DIQMTQTTSSLSASLGDRVTLSCSASQGISNYLNWYQQKPDGTVELLIFYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIK

[0324] Mouse 5F3A5D4-3 Light Chain Variable Domain Framework 1 (FR1) Sequence (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgc (amino acid) DIQMTQTTSSLSASLGDRVTLSC

[0325] Mouse 5F3A5D4-3 light chain variable domain complementarity determination region 1 (CDR1) sequence (DNA) agtgcaagtcagggcattagcaattatttaaac (amino acid) SASQGISNYLN

[0326] Mouse 5F3A5D4-3 Light Chain Variable Domain Framework 2 (FR2) Sequence (DNA) tggtatcagcagaaaccagatggaactgttgaactcctgatcttt (amino acid) WYQQKPDGTVELLIF

[0327] Mouse 5F3A5D4-3 light chain variable domain complementarity determination region 2 (CDR2) sequence (DNA) tacacatcaagtttacactca (amino acid) YTSSLHS

[0328] Mouse 5F3A5D4-3 Light Chain Variable Domain Framework 3 (FR3) Sequence (DNA) ggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt (amino acid) GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC

[0329] Mouse 5F3A5D4-3 light chain variable domain complementarity determination region 3 (CDR3) sequence (DNA) cagcagtatagtaagcttccttacacg (amino acid) QQYSKLPYT

[0330] Mouse 5F3A5D4-3 Light Chain Variable Domain Framework 4 (FR4) Sequence (DNA) ttcggaggggggaccaagctggaaataaaa (amino acid) FGGGTKLEIK

[0331] Humanized 5F3A5D4-V2 H-1.27 heavy chain variable domain sequence (DNA) caagtgcagctggtccagagcggcgccgaggtgaaaaagcctggcgccagcgtgaaggtgtcctgcaaggtgtctggcaataccttcaccgagtacaccatgcactgggtgcggcaggcccctggaaaaggcctggaatggatcggcggatttaaccccaacaacggcgtgaccaactacaac cagaagttcaagggcaaggttacactgaccgtggacaccagctcttctaccgcctacatggaactgagcagcctgagaagcgaggatacagccgtgtactattgtgccagaagatactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtgacagtgtccagc (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS

[0332] Humanized 5F3A5D4-V2 H-1.46 heavy chain variable domain sequence (DNA) caagtgcagctggtgcagagcggagccgaggtgaaaaagcccggcgcttctgtgaaggtgtcttgtaaagccagcggcaacaccttcaccgagtacaccatgcactgggtgcggcaggcccctggccagggcctggaatggatcggcggctttaatcctaacaacggcgtgacaaactacaaccagaagttcaagggcaaggttacaatcaccagagataccagcagctctaccgtgtacatggaactgagcagcctgagaagcgaggacaccgccgtgtattactgcgccagacggtactaccacagcacctacgtgttctacttcgacagctggggccagggaacactggtcacagtgtcctcc (Amino acid) QVQLVQSGAEVKKPGASVKVSCKASGNTFTEYTMHWVRQAPGQGLEWIGGFNPNNGVTNYNQKFKGKVTITRDTSSSTVYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS

[0333] Humanized 5F3A5D4-V2 H-3.15 heavy chain variable domain sequence (DNA) gaggtgcagctggtggaaagcggaggaggcctggttaagcctggaggcagcctgagactgagctgtgccgcttctggcaataccttcaccgagtacaccatgcactgggtgcggcaggcccctggcaaaggcctggaatggatcggcggcttcaaccccaacaacggcgtgacaaattacaaccagaaattcaagggcaagtttacaatcaccagagataagtctaagaacacactctatctgcaaatgaacagcctgaagtccgaggacaccgccgtgtactactgcgccagacggtactaccacagcacatacgtgttctacttcgacagctggggccagggcaccctggtcaccgtgtccagc (Amino acid) EVQLVESGGGLVKPGGSLRLSCAASGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKFTITRDKSKNTLYLQMNSLKSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS

[0334] Humanized 5F3A5D4-V2 H-4.4 heavy chain variable domain sequence (DNA) caagtgcagctgcaggagagcggacctggcctggtcaagcctggcggcaccctgagcctcacctgtgctgtttctggcaataccttcaccgagtacaccatgcactgggtgcggcagcctccaggcaaaggcctggaatggatcggcggatttaaccccaacaacggcgtgacaaattacaaccagaaattcaagggcaaggtgaccatcacagtggataagtccaagaacaccttcagcctgaagctgtctagcgtgacagccgccgacaccgccgtgtactactgcgccagaagatactatcacagcacctacgtgttctacttcgacagctggggacagggcacactggtgacagtgtccagc (amino acid) QVQLQESGPGLVKPGGTLSLTCAVSGNTFTEYTMHWVRQPPGKGLEWIGGFNPNNGVTNYNQKFKGKVTITVDKSKNTFSLKLSSVTAADTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS

[0335] Humanized 5F3A5D4-2 L-1.6 light chain variable domain sequence (DNA) gatatccagatgacacagagccctagctccctgagcgccagcgtgggcgaccgggtcaccattacatgcagcgcttctcagggcatctccaactacctgaactggtttcagcagaaacccggcaaggcccctaagctgctgatctattacaccagctctc tgcacagcggcgtgccatctagattcagcggatctggcagcggcaccgactacaccctgaccatcagctccctccagcctgaggacttcgccacctactactgtcagcaatacagcaagctgccttatacctttggcggcggaacaaaggtggaaatcaag (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWFQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKVEIK

[0336] Humanized 5F3A5D4-2 L-3.15 light chain variable domain sequence (DNA) gagatcgtgatgacccagagcccagctacacttagtgtgagtccaggtgaacgggctaccctgtcctgcagcgccagccagggcatcagcaactacctgaactggtttcagcagaaacctggccaggcccctagactgctgatctattacaccagcagcctgcacagcggcatccccgccagattcagcggcagcggctctggaacagactacaccctgacaatctctagcctgcagtctgaagattttgccgtctactactgtcagcaatacagcaagctgccttataccttcggcggcggaaccaaggtggaaattaag (amino acid) EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWFQQKPGQAPRLLIYYTSSLHSGIPARFSGSGSGTDYTLTISSLQSEDFAVYYCQQYSKLPYTFGGGTKVEIK

[0337] Humanized 5F3A5D4-2 L-4.1 light chain variable domain sequence (DNA) gatatcgtgatgacccagagcccagacagcctggcagtgagtctgggtgagcgtgctacaatcaactgcagcgccagccagggcatctccaactacctgaattggtttcagcagaaacctggccaggctcctaagctgctgatctattacaccagcagcctgcacagcggcgtgccagatagattcagcggcagcggatctggcaccgactacacactgaccatttcttctctccaggccgaggacgtggccgtctactactgtcagcaatacagcaagctgccttacacctttggcggaggcacaaaggtggaaatcaag (amino acid) DIVMTQSPDSLAVSLGERATINCSASQGISNYLNWFQQKPGQAPKLLIYYTSSLHSGVPD RFSGSGSGTDYTLTISSLQAEDVAVYYCQQYSKLPYTFGGGTKVEIK

[0338] Humanized 5F3A5D4-3 L-1.6 light chain variable domain sequence (DNA) gatatccagatgacacagagccctagctccctgagcgccagcgtgggcgaccgggtcaccattacatgcagcgcttctcagggcatctccaactacctgaactggtaccagcagaaacccggcaaggcccctaagctgctgatcttctacaccagctctc tgcacagcggcgtgccatctagattcagcggatctggcagcggcaccgactacaccctgaccatcagctccctccagcctgaggacttcgccacctactactgtcagcaatacagcaagctgccttatacctttggcggcggaacaaaggtggaaatcaag (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIFYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKVEIK

[0339] Humanized 5F3A5D4-3L-3.15 light chain variable domain sequence (DNA) gagatcgtgatgacccagagcccagctacacttagtgtgagtccaggtgaacgggctaccctgtcctgcagcgccagccagggcatcagcaactacctgaactggtaccagcagaaacctggccaggcccctagactgctgatcttctacaccagcagcctgcacagcggcatccccgccagattcagcggcagcggctctggaacagactacaccctgacaatctctagcctgcagtctgaagattttgccgtctactactgtcagcaatacagcaagctgccttataccttcggcggcggaaccaaggtggaaattaag (amino acid) EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWYQQKPGQAPRLLIFYTSSLHSGIPARFSGSGSGTDYTLTISSLQSEDFAVYYCQQYSKLPYTFGGGTKVEIK

[0340] Humanized 5F3A5D4-3 L-4.1 light chain variable domain sequence (DNA) gatatcgtgatgacccagagcccagacagcctggcagtgagtctgggtgagcgtgctacaatcaactgcagcgccagccagggcatctccaactacctgaattggtatcagcagaaacctggccaggctcctaagctgctgatcttctacaccagcagcctgcacagcggcgtgccagatagattcagcggcagcggatctggcaccgactacacactgaccatttcttctctccaggccgaggacgtggccgtctactactgtcagcaatacagcaagctgccttacacctttggcggaggcacaaaggtggaaatcaag (amino acid) DIVMTQSPDSLAVSLGERATINCSASQGISNYLNWYQQKPGQAPKLLIFYTSSLHSGVPDRFSGSGSGTDYTLTISSLQAEDVAVYYCQQYSKLPYTFGGGTKVEIK

[0341] Monoclonal antibody 8F9A5A1 Heavy chain variable region sequence H-3,4,6,10,11 atccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgttgat gacttcaagggacggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacaacctcaaaaatgaggacacgtctacatatttctgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 437)

[0342] Translation proteins in which the underlined sequence is the complementarity-determining region (CDR): IQLVQSGPELKKPGETVKISCKASG YTFTNYGMN WVKQAPGKGLKWMG WINTYTGEPTYVDDFKG RFAFSLETSATTAYLQINNLKNEDTSTYFCAR LRGIRPGPLAY WGQGTLVTVSA (Sequence ID 438)

[0343] Heavy chain variable region CDR1: YTFTNYGMN (Sequence ID 439)

[0344] Heavy chain variable region CDR2: WINTYTGEPTYVDDFKG (Sequence ID 440)

[0345] Heavy chain variable region CDR3: LRGIRPGPLAY(SEQ ID NO: 441)

[0346] Light chain variable region arrangement K-1, 2, 3, 4, 5 gaaattttgctcacccagtctccagcaatcatagctgcatctcctggggagaaggtcaccatcacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaaccaggatcctcccccaaaatatggatttatggtatatccaacctggcttct ggagttcctgctcgcttcagtggcagtgggtctgggacatctttctctttcacaatcaacagcatggaggctgaagatgttgccacttattactgtcagcaaaggagtagttacccacccacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 442)

[0347] Translation proteins in which the underlined sequence is the complementarity-determining region (CDR): EILLTQSPAIIAASPGEKVTITC SASSSVSYMN WYQQKPGSSPKIWIY GISNLAS GVPARFSGSGSGTSFSFTINSMEAEDVATYYC QQRSSYPPT FGGGTKLEIKR (Sequence ID 443)

[0348] Light chain variable region CDR1: SASSSVSYMN (Sequence ID 444)

[0349] Light chain variable region CDR2: GISNLAS (Sequence ID 445)

[0350] Light chain variable region CDR3: QQRSSYPPT (Sequence ID 446)

[0351] 8F9A4P3 Heavy Chain Variable Region Array Mouse Gtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaag ttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 335) VQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVSS(Sequence ID 1001)

[0352] IGHV1-24 * 01 V-REGION sequence human (closest matching human antibody sequence) Caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggtt ttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca (SEQ ID NO: 336) QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT(Sequence ID 1002)

[0353] Human (the closest matching human antibody sequence) IGHJ4 * 01 J-REGION arrangement tactttgactactggggccaaggaaccctggtcaccgtctcctca(SEQ ID NO: 337) YFDYWGQGTLVTVSS (Sequence ID 1003)

[0354] Humanized heavy chain variable sequence (SEQ ID NO: 1001 + SEQ ID NO: 1002 + SEQ ID NO: 1003) Humanized 8F9A4P3 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctctcctgcaaggtttcggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccaga agttcaagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 338) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (Sequence ID 1004)

[0355] Humanized heavy chain variable sequence (1004 codon-optimized version) Humanized 8F9A4P3 heavy-chain variable region sequence (codon optimized) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccaga aattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct (SEQ ID NO: 339) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (Sequence ID 1005)

[0356] Humanized heavy chain variable sequence ("modified" sequence number 1005, where modification means that certain amino acids considered critically important for binding or structure have been returned to the mouse sequence). Modified sequence 8F9A4P3 heavy-chain variable region sequence caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccaga agttcaagggcaaagtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 340) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(Sequence ID 1006)

[0357] Humanized heavy chain variable sequence (optimized for SEQ ID NO: 1006 codon) Modified humanized 8F9A4P3 heavy-chain variable region sequence (codon optimized) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct(SEQ ID NO: 341) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(SEQ ID NO: 1007)

[0358] 8F9A4P3 light chain variable region sequence, mouse gaaacaactgtgacccagtctccagcatccctgtccatggctataggagaaaaagtcaccatcagatgcataaccagcactgatattgatgatgatatgaactggtaccagcagaagccaggggaacctcctaagctccttatttcagaaggcaatactcttcgtcctggagtcccatcccgattctccagcagtggctatggtacagattttgtttttacaattgaaaacatgctctcagaagatgttgcagattactactgtttgcaaagtgataacttgcctctcacgttcggctcggggacaaagttggaaataaaacgg(SEQ ID NO: 342) ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGSGTKLEIKR (Sequence ID 1008)

[0359] Human (the closest matching human antibody sequence) IGKV5-2 * 01 V-REGION arrangement gaaacgacactcacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcaaagccagccaagacattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattt tcattattcaagaagctactactctcgttcctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgt (SEQ ID NO: 343) ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFC (Sequence ID 1009)

[0360] Human (the closest matching human antibody sequence) IGKJ4 * 02 J-REGION arrangement ctcacgttcggcggagggaccaaggtggagatcaaa(Sequence ID 344) LTFGGGTKVEIK (Sequence ID 1010)

[0361] Humanized light chain variable sequence (SEQ ID NO: 1008 + SEQ ID NO: 1009 + SEQ ID NO: 1) Humanized 8F9A4P3 light chain variable region sequence gaaacgacactcacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaaggcaatactcttcgtc ctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 345) ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (Sequence ID 1011)

[0362] Humanized light chain variable sequence (1011 codon-optimized version) Humanized 8F9A4P3 light chain variable region sequence (codon optimized) Gagacaaccctgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaacatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcttcatcatccaagagggcaacacactgcggc ctggcatccctcctagattttctggcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 346) ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (Sequence ID 1012)

[0363] Humanized light chain variable sequence ("modified" sequence number 1012, where modification means that certain amino acids considered critically important for binding or structure have been returned to the mouse sequence). Modification sequence 8F9A4P3 light chain variable region sequence gaaacgacagtgacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtc ctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 347) ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (Sequence ID 1013)

[0364] Humanized light chain variable sequence (1013 codon-optimized version) Humanized 8F9A4P3 light chain variable region sequence (codon optimized) gagacaaccgtgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaccatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcctgcttatctctgagggaaacacactgcggc ctggcatccctcctagattttccagcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 348) ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (Sequence ID 1014)

[0365] Humanized heavy and light chains connected via flexible linkers Modified humanized 8F9A4P3 sequence (codon optimized) Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgaaacgacagtgacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtcctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 349) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (Sequence ID 1015)

[0366] 8F9A5A1 Heavy Chain Variable Region Sequence atccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgttgat gacttcaagggacggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacacctcaaaaatgaggacacgtctacatatttctgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 350) IQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYVDDFKGRFAFSLETSATTAYLQINNLKNEDTSTYFCARLRGIRPGPLAYWGQGTLVTVSA (Sequence ID 1016)

[0367] IGHV7-81 * 01 V-REGION arrangement caggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctggttacagtttcaccacctatggtatgaattgggtgccacaggcccctggacaagggcttgagtggatgggatggt tcaacacctacactgggaacccaacatatgcccagggcttcacaggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcgaga (SEQ ID NO: 351) QVQLVQSGHEVKQPGASVKVSCKASGYSFTTYGMNWVPQAPGQGLEWMGWFNTYTGNPTYAQGFTGRFVFSMDTSASTAYLQISSLKAEDMAMYYCAR (Sequence ID 1017)

[0368] IGHJ4 * 03 J-REGION arrangement tactttgactactggggccaagggaccctggtcaccgtctcctca(Sequence ID 352) YFDYWGQGTLVTVSS (Sequence ID 1018)

[0369] Humanized 8F9A5A1 heavy chain variable region sequence Caggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgccacaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 353) QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1019)

[0370] Humanized 8F9A5A1 heavy chain variable region sequence (codon-optimized) caggttcagctggtgcagtctggccacgaagtgaaacagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacacctttaccaactacggcatgaactgggtgccccaggctcctggacaaggcttggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgtgttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggatatggccatgtactactgcgccagactgagaggcatcagacctggacctctggcctattggggacagggcacactggtcacagtgtcctct (SEQ ID NO: 354) QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS(Sequence ID 1020)

[0371] Modified humanized 8F9A5A1 heavy chain variable region sequence cagatccagctggtgcagtctggccccgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttg atgacttcaagggacggtttgccttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacaccgccacctattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 355) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS(Sequence ID 1021)

[0372] Modified humanized 8F9A5A1 heavy chain variable region sequence (codon optimized) cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctct(SEQ ID NO: 356) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS(SEQ ID NO: 1022)

[0373] 8F9A5A1 light chain variable region sequence gaaattttgctcacccagtctccagcaatcatagctgcatctcctggggagaaggtcaccatcacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaaccaggatcctcccccaaaatatggatttatggtatatccaacctggcttctggagttcctgctcgcttcagtggcagtgggtctgggacatctttctctttcacaatcaacagcatggaggctgaagatgttgccacttattactgtcagcaaaggagtagttacccacccacgttcggaggggggaccaagctggaaataaaacgg(SEQ ID NO: 357) EILLTQSPAIIAASPGEKVTITCSASSSVSYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR(Sequence ID 1023)

[0374] IGKV3D-15 * 02 V-REGION arrangement gaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatc tatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataataac (SEQ ID NO: 358) EIVMMQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNN(Sequence ID 1024)

[0375] IGKJ4 * 02 J-REGION arrangement ctcacgttcggcggagggaccaaggtggagatcaaa(Sequence ID 359) LTFGGGTKVEIK (Sequence ID 1025)

[0376] Humanized 8F9A5A1 light chain variable region sequence gaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctcctcatctatggtatatccaacctggcttctggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg(SEQ ID NO: 360) EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(SEQ ID NO: 1026)

[0377] Humanized 8F9A5A1 light chain variable region sequence (codon-optimized) gagatcgtgatgatgcagagccccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgctgatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccgagttcaccctgacaatctctagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 361) EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(SEQ ID NO: 1027)

[0378] Modified humanized 8F9A5A1 light chain variable region sequence gaaatagtgctgacccagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctctggatctatggtatatccaacctggcttct ggcatcccagccaggttcagtggcagtgggtctgggacaagcttcagcctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 362) EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(Sequence ID 1028)

[0379] Modified humanized 8F9A5A1 light chain variable region sequence (codon optimized) (DNA) gagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagc ggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 363) (amino acid) EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQ ID NO: 1029)

[0380] Modified humanized 8F9A5A1 scFV sequence (codon-optimized) Cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 364) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (Sequence ID 1030)

[0381] 8H5H5G4 heavy chain variable region sequence gtccagctgcaacagtctggacctgatctggtgaagcctggagacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaag ttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 365) VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS(Sequence ID 1031)

[0382] IGHV1-24 * 01 V-REGION arrangement (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggtt ttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca (SEQ ID NO: 366) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT(Sequence ID 1032)

[0383] IGHJ4 * 03 J-REGION arrangement (DNA) tactttgactactggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 367) (amino acid) YFDYWGQGTLVTVSS (Sequence ID 1033)

[0384] Humanized 8H5H5G4 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctctcctgcaaggtttccggaaacacattcactgaatacaccatgcacTgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccaga agttcaagggcAgagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtGcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 368) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (Sequence ID 1034)

[0385] Humanized 8H5H5G4 heavy chain variable region sequence (codon optimized) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccaga aattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct (SEQ ID NO: 369) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (Sequence ID 1035)

[0386] Modified humanized 8H5H5G4 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatcggaggttttaatcctaacaatggtgttactaactacaaccaga agttcaagggcaaggtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 370) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS(Sequence ID 1036)

[0387] Modified humanized 8H5H5G4 heavy chain variable region sequence (codon optimization) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccaga aattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct (SEQ ID NO: 371) (amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (Sequence ID 1037)

[0388] 8H5H5G4 light chain variable region array (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacatt caggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 372) (amino acid) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(Sequence ID 1038)

[0389] IGKV1-27 * 01 V-REGION arrangement (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcattagcaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctat gctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataacagtgcccct (SEQ ID NO: 373) (amino acid) DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP (Sequence ID 1039)

[0390] IGKJ4 * 02 J-REGION arrangement (DNA) ctcacgttcggcggagggaccaaggtggagatcaaa(Sequence No. 374) (amino acid) LTFGGGTKVEIK (Sequence ID 1040)

[0391] Humanized 8H5H5G4 light chain variable region sequence (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaacTggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacatt caggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 375) (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR (Sequence ID 1041)

[0392] Humanized 8H5H5G4 light chain variable region sequence (codon optimized) (DNA) gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcaca gcggcgtgccaagcagattttctggcagcggctctggcaccgacttcaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 376) (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(Sequence ID 1042)

[0393] Modified humanized 8H5H5G4 light chain variable region sequence (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacatt caggggtcccatctcggttcagtggcagtggatctgggacagattacactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 377) (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(Sequence ID 1043)

[0394] Modified humanized 8H5H5G4 light chain variable region sequence (codon optimization) (DNA) gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcaca gcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 378) (amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(Sequence ID 1044)

[0395] Modified humanized 8H5H5G4 scFV sequence (codon optimized) (DNA) Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 379) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR (Sequence ID 1045)

[0396] Human IgG1 heavy chain constant region sequence: (Express the two plasmids together to create a complete antibody pair with either the kappa or lambda constant region.) (DNA) (amino acid) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 1046)

[0397] Human IgG double-chain constant region sequence: (Express two plasmids together to create a complete antibody pair containing either the kappa or lambda constant region.) (DNA) (query number 381) (amino acid) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 1047)

[0398] Human kappa light chain constant region sequence (DNA) aggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtg tcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 382) (amino acid) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 1048)

[0399] Human lambda light chain constant region sequence (DNA) ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagacc accacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatag (SEQ ID NO: 383) (amino acid) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 1049)

[0400] Human IgG1 Fc region sequence (fused to scFv for homodimerization) (DNA) gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 384) (amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK * (SEQ ID NO: 1050)

[0401] Human IgG2 Fc region sequence (DNA) gagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag (SEQ ID NO: 385) (Amino acid) ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK * (Sequence ID 1051)

[0402] In another aspect of the present invention, immune cells engineered to express CAR are administered to patients diagnosed with cancer or cancer metastasis, or patients at risk of developing cancer or cancer metastasis, and the immune cells are also engineered to express anti-NME7 antibodies or antibody fragments, which may be expressed from an inducible promoter. In one aspect, CAR is MUC1 * It is induced by an antibody fragment. In one example, the CAR is huMNC2-CAR44. In one embodiment, an anti-NME7 antibody or antibody fragment binds to an NME peptide listed above in sections “Peptides homologous to A1, A2, B1, B2, or B3 peptides” and “Extended peptides homologous to A1, A2, B1, B2, or B3 peptides”. In another embodiment, 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 embodiment, the antibody, antibody fragment, or antibody mimetic binds to an NME7 peptide containing the B3 peptide. In one embodiment of the present invention, the anti-NME7 antibody, antibody fragment, or antibody mimetic contains a sequence derived from the variable domain of the anti-NME7 antibody 8F9A4A3, 8F9A5A1, or 8H5H5G4.

[0403] Such antibodies may be human antibodies or humanized antibodies. Such antibodies may be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or antibody mimics such as protein scaffolds that provide a recognition region for binding to a specific target. As will be understood by those skilled in the art, antibodies may be of non-human origin, human, or humanized antibodies. Methods for humanizing antibodies include fusing all or part of a mouse variable region to the V and J regions of a nearest-neighbor-match human antibody sequence, such as the sequences listed as SEQ ID NOs: 1001-1045. Complete antibodies, rather than single-chain constructs, can also be produced. For example, a heavy-chain variable mouse sequence is fusing to human V and J regions, and then to IgG1, IgG2, or IgG3 of a human heavy-chain constant region. Similarly, a light-chain variable mouse sequence is fusing to human V and J regions, and then to IgG1, IgG2, or IgG3 of a human kappa or lambda constant region. Plasmids are then expressed together and bound to form complete antibodies (SEQ ID NOs: 1047-1051).

[0404] In another aspect of the present invention, the small molecule is NME7, NME7 AB Alternatively, they are anticancer agents selected for their ability to inhibit the tumorigenic effects of NME7-X1. For example, high-throughput screening identifies small molecules that treat cancer. In multi-well plates, the small molecules are identified when cancer cells in which NME7-X1 is detected. AB It is added separately to wells cultured in a medium containing [the substance]. If the small molecule reduces the amount of cells that become suspension and / or reduces the expression of metastasis markers or pluripotent stem cell markers such as CXCR4, CHD1, then that small molecule is a candidate for an anticancer drug. Another way to identify small molecules that are anticancer drugs is NME7, NME7 AB Alternatively, one could select small molecules that bind to NME7-X1 or suppress the expression of NME7 species. Another high-throughput screening method is MUC1 * NME7 to PSMGFR peptide in the extracellular domain AB The goal is to select small molecules that inhibit the binding of these molecules, and these small molecules are anticancer drugs.

[0405] NPE7 AB The sequence of NME7-X1 is such that NME7-X1 is NME7 AB It differs only in that it lacks some of the N-terminal sequences it possesses. The experiment was conducted using NME7. AB The inventors have shown that there are seven naturally occurring NMEs that are almost identical to NMEs, and they have identified them as NMEs. AB This is called a variant. Antibodies that bind to NME7-X1 will not recognize NME7 unless there is a higher-order structural difference that the antibody can distinguish. AB It can also bind to naturally occurring species that mimic it. Therefore, NME7 AB When it is desired to suppress NME7-X1 rather than a specific species, or vice versa, siRNA, antisense nucleic acids, or gene editing technologies can be used to suppress the expression of one species while not suppressing others.

[0406] For example, anticancer drugs include NME7, NME7-X1, or NME7 AB These nucleic acids directly or indirectly suppress the specific expression of various species. Such nucleic acids may be siRNA, RNAi, or antisense nucleic acids that directly suppress the NME7 species. In another aspect of the present invention, nucleic acids can indirectly suppress the NME7 species, for example, by altering the expression of molecules that regulate them. For example, the super-enhancer BRD4 suppresses the expression of NME7. Therefore, effective therapeutic agents for the treatment or prevention of cancer are agents that increase the expression of BRD4. Effective therapeutic agents may be agents that increase the expression of the BRD4 cofactor, JMJD6.

[0407] NPE7 AB Alternatively, NME7-X1 derived peptides or whole proteins can be used in animals to generate anti-NME7 and anti-NME7-X1 antibodies, which the inventors have demonstrated to suppress 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 that treat or prevent cancer or inhibit the migration of cancer cells to metastatic cancer cells. NME7 peptides or proteins, in the recipient, produce anti-NME7 and anti-NME7 ABIt is administered to humans as a type of vaccine to stimulate the production of anti-NME7-X1 antibodies. The results shown in Figures 12 and 13 are derived from NME7, particularly NME7-X1 or NME7 AB This suggests that immunizing humans with a group of peptides in a sequence may result in a more effective vaccine than immunization with a single peptide. The peptide or protein may be further conjugated to a carrier protein or other adjuvant, which are known to those skilled in the art to assist in stimulating the immune response.

[0408] The NME7 peptide located outside the DM10 domain is preferred for generating antibodies for the treatment or prevention of cancer. Peptides that can be administered to patients for the prevention of cancer or metastasis include the peptide sequences listed in Figures 6-9. A1, A2, B1, B2, and B3 are NME7 peptides. AB Examples of peptides that bind to NME7-X1 and produce antibodies administered to patients for the treatment or prevention of cancer are also included. The present invention is not limited to peptides with precise sequences such as the naturally occurring sequences in NME7 or NME7-X1. As is known to those skilled in the art, substitutions of several amino acids in a peptide sequence can still produce antibodies that specifically recognize the native protein sequence. The present invention is not intended to be limited to the peptides demonstrated herein that inhibit cancer growth or inhibit the normal migration of 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 equivalent to or more effective than the peptides demonstrated herein.

[0409] Human NME7 AB Or includes part of NME7-X1, or NME7 AB Alternatively, chimeric antigen receptor molecules containing antibody fragments that bind to NME7-X1 are anticancer drugs administered to patients for the treatment or prevention of cancer or cancer metastasis.

[0410] In one example, the recognition unit or variable region of an anti-NME7 antibody is fused to a T cell molecule using a technique known as CAR (chimeric antigen receptor) technology or CAR T technology. A notable feature of an antibody or fragment 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 cancer-promoting targets. In one example, the target is MUC1 * This is the PSMGFR region.

[0411] Antibodies, antibody fragments, or single-chain antibodies can be manipulated into chimeric molecules containing chimeric antigen receptors, also known as CARs, which are then transfected or transfected into immune system cells such as T cells and administered to patients. Humanized antibodies or antibody fragments, typically scFvs, contain many of the extracellular domains of CARs. The antibody fragment is biochemically fused to immune system signaling molecules such as CD8 as a transmembrane domain, cytoplasmic signaling motifs such as T cell receptor signaling molecules also called activating domains, or, but not limited to, CD3 zeta, CD28, 41bb, OX40, etc. The CAR can be transfected into T cells or other cells, preferably immune system cells, and administered to patients. Here, the inventors have found that the extracellular portion contains anti-NME7, anti-NME7 AB Alternatively, the description may include a CAR comprising an anti-NME7-X1 antibody, an antibody fragment or single chain, or an scFv antibody fragment. In preferred embodiments, the antibody or antibody fragment is a human or humanized antibody.

[0412] Effective anti-NME7 or anti-NME7-X1 antibodies, or fragments, are derived from natural NME7, NME7 AB Alternatively, it has the ability to bind to NME7-X1. In fact, the parent antibody from which the extracellular domain of CAR is manipulated is NME7, NME7 ABAlternatively, it is produced by immunizing animals with an NME7-X1 derived peptide. In one aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 1-376. In another aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 92-376. In yet another aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 125-376. In yet another aspect of the present invention, the immunizing peptide is composed of the sequences listed in Figures 6-8. In yet another aspect of the present invention, the immunizing peptide is composed of the sequences listed in Figure 9. Alternatively, the parent antibody or antibody fragment may be selected from an antibody library or pool of natural, synthetic, or any other fragment, and they may be NME7, NME7 AB Alternatively, the selection is based on the ability to bind to NME7-X1, the peptides listed in Figures 6-8, or the peptides listed in Figure 9.

[0413] The targeting portion of a CAR does not need to be an antibody or antibody fragment. Here, we describe a CAR in which the extracellular domain contains an NME7 fragment. NME7-derived peptides are manipulated in different types of CARs in which the targeting portion of the extracellular domain is a protein fragment or peptide, rather than an antibody or antibody fragment. Peptide CARs are transfected or transfected into immune system cells, typically T cells. NME7 fragments or NME7-derived peptides bind to their homologous binding partners, but intact NME7, NME7 AB The selection is based on the ability to function as, or not function like, NME7-X1, and not confer oncogenic activity. NME7 fragments or NME7-derived peptides are biochemically fused to cytoplasmic signaling motifs such as CD8 as a transmembrane domain, a T cell receptor signaling molecule also called an activating domain, or to immune system signaling molecules such as, but not limited to, CD3 zeta, CD28, 41bb, OX40, and co-stimulatory domains.

[0414] 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 Figures 6-9. Experimental results show that NME7 or NME7, NME7 as the targeting portion of a chimeric antigen receptor (CAR) for engineered immunotherapy agents. AB Alternatively, in the case of a strategy using fragments of NME7-X1, NME7 AB Alternatively, it could indicate that a fairly large fragment of NME7-X1 may be more effective than shorter peptides, e.g., peptides less than 15 amino acids in length. Or, each of them could be a different NME7. AB A group of CARs containing the derived peptide can be collectively transduced or transduced into immune system cells and administered to patients for the treatment or prevention of cancer. Experiments shown in Figures 12 and 13 support the validity of this approach.

[0415] CARs containing the NME7 fragment in their extracellular domain are transduced or transduced into immune system cells, typically T cells, and administered to patients for the treatment or prevention of cancer. In one embodiment, cancer is MUC1 * It is a positive cancer. In another context, the cancer is metastatic cancer.

[0416] Drugs that inhibit the enzyme that cleaves NME7 can be used to treat or prevent cancer. Some forms of NME7 are sequestered within cells and therefore not secreted from cells, in which case they may act as growth factors to promote cancer. Full-length NME7 is 42 kDa. However, the inventors have developed recombinant NME7 lacking the DM10 domain and generated by the inventors. AB We found that seven types of NME7, approximately 33 kDa in size and virtually identical to the aforementioned NME7, are secreted from cancer cells and stem cells. These seven types, approximately 33 kDa and another approximately 25 kDa, may be cleavage products removed by the use of drugs that inhibit NME7 cleavage.

[0417] High levels of NME7 in patient samples, including NME7 species with a coherence of approximately 33 kDa (the inventors identified this as NME7 AB The detection of MUC1 (referred to as MUC1), or NME7-X1, is an indicator of the presence of cancer or its progression to a more malignant or metastatic state. The inventors believe that in both the early stages, naive stem cells and cancer cells, particularly MUC1 * We found that positive cancer cells express high levels of NME7 and NME7-X1, both approximately 33 kDa and lacking the DM10 domain.

[0418] NME7-X1 was recently listed in protein databases as a theoretically selective isoform of NME7. However, it has not been detected in tissues or cells. We designed primers to distinguish NME7-X1 from NME7 by PCR. 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 hepatocytes, 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 times more highly in HEK293 fetal hepatocytes than in fibroblasts or stem cells; therefore, NME7-X1 is expected to be elevated in liver cancer. NME7b is expressed 17 to 25 times more highly in prostate cancer cells than in stem cells.

[0419] High levels of NME7 in patient samples indicate that the patient has cancer or is at risk of developing cancer. NME7 levels can be measured or evaluated by PCR, hybridization schemes, cycling probe techniques, FISH, immunocytochemistry, IHC, Western blotting, immunoprecipitation, sandwich analysis, ELISA assays, etc. Patient samples may include liquid samples, blood samples, breast milk, urine, cells, liquid biopsies, biopsies, etc. In patients diagnosed with cancer, high levels of NME7 are an indicator of increased metastatic potential. High levels of NME7-X1 are an indicator of prostate cancer. The antibodies of this invention are used to detect and differentiate NME7 and are used as a diagnostic tool.

[0420] Since adult human cells and tissues do not express or secrete NME7 at significant levels, an effective method for diagnosing cancer, or a more aggressive or metastatic form, or a shift to a more aggressive form, is to measure NME7 levels in samples from patients, from cell or tissue collections, or from cultured cells, compared to NME7 levels in healthy samples and / or levels of NME7 known to be present in healthy adult cells or tissues. Elevated levels of NME7 indicate the presence of cancer, the presence of metastatic cancer, or the initiation of metastasis. Elevated levels of NME7 also indicate MUC1 *This indicates positive cancer. Samples assayed for the presence of NME7 may be cell collections from a patient, which may be cultured cell lines, bodily fluids, blood samples, tissue specimens, or biopsy materials. Therefore, a diagnostic assay to detect the presence or progression of cancer may include 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 levels of NME7 or nucleic acids encoding NME7; 3) comparing the measured levels of NME7 protein or NME7-coding nucleic acids in the test sample to levels in a control patient or control cells; 4) identifying that the levels of NME7 or nucleic acids encoding NME7 are elevated compared to the control; and 5) concluding that cancer is progressing if the donor of the test sample has cancer, or if the control to which the test sample is compared is from a donor previously diagnosed with cancer.

[0421] In this assay, the control sample against which the test sample is compared may be non-cancerous cells, cultured cells, a sample from a healthy donor, a non-cancerous sample from a donor, or a sample from the donor of the test sample that is a control sample taken from the donor at a previous point in time. Such sample sources may be any specimen taken from the patient being tested for the presence or progression of cancer, including body fluids, cerebrospinal fluid, bone marrow samples, blood, tissues, cells, biopsy tissue or cells, or cultured cells derived from patient cells. The sample source against which the test sample is compared may be body fluids, cerebrospinal fluid, bone marrow samples, blood, tissues, cells, biopsy tissue or cells, or cultured cells from a healthy donor or a patient being tested from which the sample was obtained at a previous point in time. The level of measurement against which the test sample is compared may be derived from previously recorded data and accumulated in a list for comparison to the test sample.

[0422] Theranostics Patients diagnosed with high levels of the NME7 protein or the nucleic acid encoding NME7 are subsequently treated with therapies that suppress NME7 expression, inhibit NME7 cleavage, or inhibit NME7 binding to its targets (such interactions promote cancer). Key targets of NME7 or cleavage products of NME7 include MUC1. * NME7 is MUC1 * It binds to the extracellular domain and dimerizes it. Therefore, patients diagnosed with high levels of NME7 would benefit from treatment with therapies that suppress NME7 and / or the cleavage-type dimerization of MUC1, which consists of some or all of the PSMGFR sequence in its extracellular domain. Thus, evaluating the suitability of cancer treatment and the administration of an effective dose of the therapy 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 products, NME7-coding nucleic acids, 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 more aggressive or metastatic cancer; 4) administering an effective dose of the therapy to the patient that suppresses NME7 expression, suppresses NME7 cleavage, or suppresses the binding of NME7 to its target, and / or suppresses MUC1 expression, MUC1 * Suppresses MUC1 cleavage to or targets of MUC1 * A step of administering to the patient an effective amount of a therapeutic agent that inhibits the binding of MUC1. In a preferred embodiment, the therapeutic agent that inhibits the binding of NME7 to its target is MUC1 * It suppresses its interaction with. In a more preferred embodiment, it is MUC1 substantially composed of PSMGFR sequences. * It suppresses its interaction with the extracellular domain. In a preferred embodiment, MUC1 to its target * Therapeutic drugs that inhibit binding are MUC1 * The interaction between and NME7 is suppressed. In a more preferred embodiment, MUC1 *Therapeutic drugs that suppress the interaction between and NME7 AB MUC1 to the portion of NME7 that is essentially composed of the sequence * It inhibits the binding.

[0423] Chemically modified peptides Polypeptides or antibody therapeutics have problems with short circulating half-lives, proteolysis, and low solubility. To improve the pharmacokinetic and pharmacodynamic properties of the biopharmaceuticals of the present invention, methods such as manipulating amino acid sequences may reduce or increase immunogenicity and reduce protein cleavage; fusion or binding of peptides to immunoglobulins such as albumin and serum proteins may be performed; incorporation of the peptides and biopharmaceuticals such as antibodies of the present invention into drug delivery carriers may also be performed for protection and sustained release; and binding to natural or synthetic polymers may also be considered. In particular, for binding to synthetic polymers, pegylation or acylation such as N-acylation and S-acylation may also be considered.

[0424] nucleic acid construct Expression vectors containing the nucleic acid molecule of the present invention, as described herein, are also provided, in which the nucleic acid molecule is operably ligated to an expression regulatory sequence. Furthermore, a host-vector system is provided for the production of polypeptides containing the expression vector of the present invention, introduced into a host cell suitable for polypeptide expression. Suitable host cells may be bacterial cells such as Escherichia coli (E. coli), yeast cells such as Pichia pastrius, insect cells such as armyworms, or mammalian cells such as COS, HEK, or CHO cells.

[0425] The present invention also provides a method for producing polypeptides of the present invention by growing cells of the host-vector system described herein under conditions that allow polypeptide production, and a method for recovering the polypeptides thus produced. Polypeptides useful for carrying out the present invention can be prepared by expression in a prokaryotic or eukaryotic expression system.

[0426] Recombinant genes are expressed, and polypeptides are purified using various methods. The genes can be subcloned into bacterial expression vectors, such as pZErO, though this is not limited to these methods.

[0427] Polypeptides can be purified by any technique that allows for the subsequent formation of stable, biologically active proteins. For example, but not limited to, factors may be recovered from cells either as soluble proteins or as inclusion bodies, from which they are quantitatively extracted with 8M guanidium hydrochloride and dialyzed. To further purify the factors, numerous purification methods are used, but not limited to, conventional ion-exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, or gel filtration.

[0428] As used herein, polypeptides include functionally equivalent molecules in which an amino acid residue is substituted for a residue in the sequence, resulting in a silent or conservative change. For example, one or more amino acid residues in the sequence can be substituted with another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent or conservative change. The substitution of an amino acid in the sequence may 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. Neutral polarity 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. Furthermore, the scope of the present invention includes proteins or fragments or derivatives thereof exhibiting the same or similar biological activity, and derivatives that are modified differently during or after translation, for example, by glycosylation, protein cleavage, or linkage to antibody molecules or other cellular ligands.

[0429] Any method known to those skilled in the art for inserting DNA fragments into vectors may be used to construct expression vectors encoding the polypeptide of the present invention using appropriate transcription / translation control signals and protein-coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). The expression of the nucleic acid sequence encoding the polypeptide of the present invention may be regulated by a second nucleic acid sequence, thereby the polypeptide being expressed in a host transformed with a recombinant DNA molecule. For example, the expression of the polypeptide described herein may be regulated by any promoter / enhancer sequence known in the art. Promoters that may be used to control polypeptide expression include, but are not limited to, the following: long-terminal repeat sequences 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 included in the 3' long-terminal repeat of Roussarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:144-1445); and the regulatory sequence of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); Prokaryotic expression vectors such as β-lactamase promoters (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or tac promoters (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. USA 75:3727-3731).See also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94 (80:21-25); promoter sequences from yeast or other fungi, e.g., the Gal 4 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerate kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional regulatory regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene regulatory region 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); insulin gene regulatory region active in pancreatic β cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene regulatory regions 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), mouse mammary tumor virus regulatory regions active in testes, mammary glands, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), Sendai virus, lentivirus, albumin gene regulatory regions active in liver (Pinkert et al., 1987, Genes and Devel.1:268-276), alpha-fetoprotein gene regulatory regions active in liver (Krumlauf et al.) al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); Alpha-1 antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel.1:161-171), the β-globin gene regulatory region 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 active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene regulatory region active in skeletal muscle (Shani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0430] Accordingly, the present invention provides an expression vector, which can be replicated in a bacterial or eukaryotic host, comprising a nucleic acid encoding a polypeptide described herein, to be used to introduce a gene 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, the biologically active form includes a form that can bind to a relevant receptor, induce differentiation function, and / or influence the expression type of a cell expressing the receptor.

[0431] Expression vectors containing nucleic acid inserts can be identified by at least three common methods, though not limited to them: (a) DNA-DNA hybridization, (b) the presence or absence of “marker” gene function, and (c) expression of the insert sequence. In the first method, the presence of heterologous nucleic acid inserted into an 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 specific “marker” gene function (e.g., thymidine kinase activity, antibiotic resistance, transformed phenotype, occlusion formation in baculoviruses) caused by the insertion of heterologous nucleic acid sequences in the vector. For example, if an efl nucleic acid sequence is inserted into a marker gene sequence in a vector, recombinants containing the insert can be identified by the absence of marker gene function. In the third method, recombinant expression vectors can be identified by assays of heterologous nucleic acid products expressed by the recombinant construct. Such assays may be based on the physical or functional properties of the nucleic acid product of interest, for example, by binding of a ligand to a receptor or its moiety that can be tagged with a detectable antibody or its moiety, or by binding to an antibody produced against the protein or its moiety of interest.

[0432] Polypeptides, particularly the modified polypeptides of the present invention, can be expressed in host cells transiently, constitutively, or permanently.

[0433] Effective amounts useful for treating the diseases or disorders described in the present invention can be determined by methods known to those skilled in the art (e.g., Fingl, et al., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co, New York, pp. 1-46 (1975)). Pharmaceutical compositions for use according to the present invention comprise the polypeptide described above in a pharmaceutically acceptable liquid, solid, or semi-solid carrier, which is linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and / or incorporated into liposomes, microcapsules, and sustained-release formulations before in vivo administration. For example, a pharmaceutical composition may comprise the polypeptide in an aqueous solution such as sterile water, physiological saline, phosphate buffer, or dextrose solution. Alternatively, the active agent may be contained in a solid (e.g., wax) or semi-solid (e.g., gelatin) formulation that can be implanted into a patient requiring such treatment. The route of administration may be any method of administration known in the art, but is not limited to, intravenous, subarachnoid, subcutaneous, intrauterine, injection into diseased tissue, intraarterial, intranasal, oral, or via an implantable device.

[0434] 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 areas of the nervous system, intravenous or subarachnoid administration of the agent may be preferable. In some cases, an implantable tablet containing the active agent may be placed in or near the injured area. Suitable implantable tablets include, but are not limited to, gel foams, waxes, sprays, or particulate-based implantable tablets.

[0435] The present invention also provides pharmaceutical compositions comprising polypeptides described herein in a pharmaceutically acceptable carrier. The compositions may be administered systemically or topically. Any method of administration known in the art may be used, but are not limited to, intravenous, subarachnoid, intra-arterial, intranasal, oral, subcutaneous, intraperitoneal, local injection, or surgical implantation. Sustained-release formulations are also provided.

[0436] gene therapy Gene therapy refers to a therapy performed by administering expressed or potentially expressible nucleic acids to a target. In this embodiment of the present invention, the nucleic acids produce their encoded proteins that mediate the therapeutic effect.

[0437] Any method for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are described below.

[0438] 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). Commonly known methods in the field 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).

[0439] Nucleic acid delivery to a patient can be direct or indirect. In the direct case, the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector. In the indirect case, cells are first transformed in vitro with the nucleic acid and then transplanted into the patient. These two methods are known as in vivo or ex vivo gene therapy, respectively.

[0440] In certain embodiments, nucleic acid sequences are administered directly in vivo, where they are expressed and produce an encoded product. This can be achieved by any of the numerous methods known in the art, for example, by constructing them as part of a suitable nucleic acid expression vector and administering them so that they become intracellular; for example, by infection using a defective or attenuated retrovirus or other viral vector; or by direct injection of naked DNA; or by coating them with lipids or cell surface receptors or gene transdermal agents, encapsulating them in liposomes, microparticles or microcapsules, or by ligating them to a ligand exposed to receptor-mediated endocytosis and administering them (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (this can be used to target cell types that express receptors in particular), and so on. In another embodiment, a nucleic acid-ligand complex is formed, where the ligand contains a fusionable viral peptide that disrupts endosomes, allowing the nucleic acid to avoid lysosomal degradation. In another embodiment, nucleic acids can be in vivo targeted for cell-specific uptake and expression by targeting specific receptors. Alternatively, nucleic acids can be introduced into cells and incorporated into 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)).

[0441] In certain embodiments, viral vectors containing nucleic acid sequences encoding polypeptides are used. The nucleic acid sequences encoding polypeptides used in gene therapy are cloned into one or more vectors, which facilitate the 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 may be used. Retroviral vectors contain components necessary for the precise packaging of the viral genome and its integration into host cell DNA.

[0442] Adenoviruses are particularly attractive carriers for delivering genes to the airway epithelium because they spontaneously infect the airway epithelium, causing mild illness. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. In addition, adeno-associated viruses (AAVs) have also been proposed for use in gene therapy.

[0443] Another approach to gene therapy involves transferring genes into cells in tissue culture by methods such as electroporation, lipofectin, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method involves transferring a selectable marker into the cells. The cells are then selectively treated to isolate those that have taken up and expressed the transgene. These cells are then delivered to the patient.

[0444] 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, but is 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 in the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The techniques should provide stable introduction of the nucleic acid into the cells, thereby allowing the nucleic acid to be expressed by the cells, and preferably heritable, and expressed by the offspring of those cells.

[0445] Cells into which nucleic acids can be introduced for gene therapy purposes include, but are not limited to, any desired available cell type, including, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes; and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, obtained from sources such as bone marrow, umbilical cord blood, peripheral blood, and fetal liver.

[0446] In a preferred embodiment, the cells used in gene therapy are derived from the patient's own body.

[0447] In embodiments where recombinant cells are used in gene therapy, nucleic acid sequences encoding polypeptides are introduced into cells so that they can be expressed by the cells or their offspring, and the recombinant cells are then administered in vivo for 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 probably be used according to this embodiment of the invention.

[0448] In certain embodiments, the nucleic acid introduced for gene therapy purposes includes an inducible promoter operably linked to the coding region, thereby allowing the expression of the nucleic acid to be controlled by modulating the presence or absence of a suitable transcription inducer.

[0449] therapeutic composition Formulations of therapeutic compounds are generally known in the art, and one can conveniently refer to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, approximately 0.05 ng to approximately 20 mg per kg of body weight may be administered daily. The administration plan may be adjusted to obtain the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the criticality of the treatment situation. The active compound may be administered by oral, intravenous (if water-soluble), intramuscular, subcutaneous, intranasal, intraocular, intradermal, or suppository routes or by transplantation (e.g., by using sustained-release molecules via the intraperitoneal route, or by using cells, e.g., monocytes or dendritic cells sensitized in vitro and adopted into the recipient). Depending on the route of administration, the peptide may need to be coated with a substance to protect it from the action of enzymes, acids and other natural conditions that can inactivate the component.

[0450] For example, the low lipophilicity of peptides allows them to be destroyed in the gastrointestinal tract by enzymes that can cleave peptide bonds, or in the stomach by acid hydrolysis. To administer peptides by means other than parenteral administration, the peptides are coated with a substance that prevents their inactivation, or are administered together with such a substance. For example, peptides may be administered in an adjuvant, co-administered with an enzyme inhibitor, or in liposomes. Adjuvants as intended 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 trazilol. Liposomes include water-in-oil-in-water CGF emulsions, as well as conventional liposomes.

[0451] The active compound may also be administered parenterally or intraperitoneally. Dispersants may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0452] Suitable dosage forms for injectable use include sterile aqueous solutions (if soluble) or dispersions, and sterile powders for immediate formulation with sterile injection solutions or dispersants. In all cases, the form must be sterile and fluid enough to be readily injectable. It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained by the use of coatings such as lecithin to maintain the particle size required in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents (e.g., chlorobutanol, phenol, sorbic acid, thiomersal, etc.). In many cases, it is preferable to include an isotonic agent (e.g., sucrose or sodium chloride). Sustained absorption of injectable compositions can be achieved by using absorption retarders, such as aluminum monostearate and gelatin, in the composition.

[0453] Sterile injectable solutions are prepared by incorporating the required amount of active compound in a suitable solvent along with various other components listed above, followed by sterile filtration as necessary. Generally, dispersants are prepared by incorporating various sterile active ingredients into a sterile carrier containing a basic dispersion medium and other necessary components selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a mixed powder of the active ingredient derived from the pre-sterile filtered solution and any additional desired components.

[0454] When the peptide is adequately protected as described above, the active compound is administered orally, for example, with an inert diluent or an assimilated food carrier, or it is encapsulated in a hard or soft-shell gelatin capsule, or it is compressed into a tablet, or it is directly incorporated into a food product. For oral therapeutic administration, the active compound is incorporated with excipients and can be used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations should contain at least 1% by weight of the active compound. The percentage of the composition and formulation may, of course, vary and may conveniently be between about 5% and about 80% by weight. The amount of the active compound in such a therapeutically useful composition is such that an appropriate dose is obtained. Preferred compositions or formulations according to the present invention are prepared such that the oral dose unit formulation contains between about 0.1 μg and 2000 mg of the active ingredient.

[0455] Tablets, pills, capsules, etc. may also contain: binders such as tragacanth gum, acacia gum, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint, wintergreen oil, or cherry flavorings. If the dosage unit is a capsule, it may contain a liquid carrier in addition to the above types of substances. Various other substances may be present as coating agents or for other reasons to alter 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 propylparaben as preservatives, and flavorings such as cherry or orange flavoring. Of course, any substance used in the preparation of any dosage unit must be pharmaceutically pure and substantially non-toxic in the amount used. In addition, active compounds may be incorporated into sustained-release formulations and formulations.

[0456] Delivery system Various delivery systems are known and can be used to administer the compounds of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-dependent endocytosis, nucleic acid constructs as part of retroviruses or other vectors. Methods of delivery are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example, by injection or bolus injection, by absorption through epithelium or the surface of the skin or mucous membrane (e.g., oral mucosa, rectal and intestinal mucosa), or together with other bioactive substances. Administration may be systemic or topical. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the present invention into the central nervous system by any preferred route, including intracerebroventricular injection and subarachnoid injection. Intracerebroventricular injection may be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir such as an Omaya reservoir. For example, intrapulmonary administration can also be employed by using a formulation containing an inhaler or nebulizer and an aerosolizing agent.

[0457] In certain embodiments, it may be desirable to administer the pharmaceutical compound or composition of the present invention topically to an area requiring treatment; this can be achieved, for example, by injection, by catheter, by suppository, or by implant, in conjunction with, for example, local injection during surgery, local application, or postoperative wound dressings. The implant may be a porous, non-porous, or gelatinous material containing a membrane such as a thyrustic membrane, or fibers. Preferably, when administering a protein containing the antibody or peptide of the present invention, care must be taken to use a material that the protein does 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 by a controlled-release system. In one embodiment, a pump may be used. In another embodiment, a polymer material may be used. In yet another embodiment, the controlled-release system may be positioned near the therapeutic target, thereby requiring only a very small amount for systemic administration.

[0458] Sequence listing free text For the use of nucleotide abbreviations other than a, g, c, and t, they shall be in accordance with the rules set forth in WIPO Standard ST.25, Supplement 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.

[0459] 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 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 KVSAGNGGS LSYTNPAVAA ASANL (SEQ ID NO: 1) represents the full-length MUC1 receptor (mucin 1 precursor, Genbank accession number 15941).

[0460] MTPGTQSPFFLLLLLTVLT(Sequence ID 2) MTPGTQSPFFLLLLLTVLT VVTA(Sequence ID 3) MTPGTQSPFFLLLLLTVLT VVTG (Sequence ID 4) Sequence IDs 2, 3, and 4 are N-terminal MUC-1 signaling sequences for orienting the MUC1 receptor and its truncated isoform to the cell membrane surface. As indicated by variants in Sequence IDs 2, 3, and 4, up to three amino acid residues may be deleted at the C-terminus.

[0461] GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO: 5) represents a truncated MUC1 receptor isoform having native PSMGFR at its N-terminus, and includes the transmembrane and cytoplasmic sequences of the full-length MUC1 receptor.

[0462] GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6) represents the extracellular domain of the native primary sequence of the MUC1 receptor (an example of nat-PSMGFR-"PSMGFR").

[0463] TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 7) represents the extracellular domain of the native primary sequence of the MUC1 growth factor receptor (an example of nat-PSMGFR-"PSMGFR"), with a single amino acid deletion at the N-terminus of SEQ ID NO: 6.

[0464] GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (Sequence ID 8) represents the extracellular domain of the “SPY” functional variant of the native primary sequence of the MUC1 growth factor receptor, which has enhanced stability (an example of var-PSMGFR-“PSMGFR”.

[0465] TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 9) represents the extracellular domain of the "SPY" functional variant of the MUC1 growth factor receptor with enhanced stability, which has a single amino acid deletion at the C-terminus of SEQ ID NO: 8 (an example of var-PSMGFR- "PSMGFR").

[0466] tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacctaccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggcagcagcctctcttacacaaacccagcagtggcagccgcttctgccaacttg (SEQ ID NO: 10) represents the nucleotide sequence of the MUC1 cytoplasmic domain.

[0467] CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (Sequence ID 11) represents the amino acid sequence of the MUC1 cytoplasmic domain.

[0468] (Sequence ID 12) represents the NME7 nucleotide sequence (NME7: GENBANK acceptance number AB209049).

[0469] DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMYRK (Sequence ID 13) represents the NME7 amino acid sequence (NME7: GENBANK acceptance number AB209049).

[0470] (Sequence ID 14) represents the NM23-H1 nucleotide sequence (NM23-H1: GENBANK acceptance number AF487339).

[0471] MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE (Sequence ID 15) NM23-H1 represents the amino acid sequence (NM23-H1: GENBANK acceptance number AF487339).

[0472] (Sequence ID 16) represents the NM23-H1 S120G mutant nucleotide sequence (NM23-H1: GENBANK acceptance number AF487339).

[0473] MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE (Sequence ID 17) represents the NM23-H1 S120G mutant amino acid sequence (NM23-H1: GENBANK acceptance number AF487339).

[0474] atggccaacctggagcgcaccttcatcgcatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatcaagcgcttcgagcagaagggattccgcctcgtggccatgaagttcctccgggcc tctgaagaacacctgaagcagcactacattgacctgaaagaccgaccattcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtggtgaagaca ggccgagtgatgcttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggttggcaggaacatcattcatggcagtgattcagtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgactacaagtcttgtgctcatgactgggtctatgaataa (Sequence ID 18) represents the NM23-H2 nucleotide sequence (NM23-H2: GENBANK acceptance number AK313448).

[0475] MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (Sequence ID 19) represents the amino acid sequence of NM23-H2 (NM23-H2: GENBANK acceptance number AK313448).

[0476] Human NM23-H7-2 sequence optimized for Escherichia coli (E. coli) expression: (DNA) (amino acid) MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN (Sequence ID 21)

[0477] Human NME7-A: (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctat tattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcat tagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (SEQ ID NO: 22) (amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF(Sequence ID 23)

[0478] Human NME7-A1: (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgct ttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagag atgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaat gcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQ ID NO: 24) (amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT(Sequence ID 25)

[0479] Human NME7-A2: (DNA) (Sequence ID 26) (amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF (Sequence ID 27)

[0480] Human NME7-A3: (...

Claims

1. An NME7-specific antibody or fragment thereof that binds to the NME7 B3 peptide of SEQ ID NO: 145 or SEQ ID NO:

169.

2. The antibody according to claim 1, which is a monoclonal antibody.

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

4. The antibody according to claim 1, which is linked to an antibody-drug conjugate.

5. The antibody according to claim 1, wherein the drug is linked to a toxin or protoxin.

6. An isolated nucleic acid encoding a monoclonal antibody as described in claim 2.

7. An isolated hybridoma expressing the monoclonal antibody described in claim 2.

8. NME7 ＡＢ Alternatively, the antibody according to claim 1, which specifically binds to NME7-X1 but does not specifically bind to NME1.

9. NME7 ＡＢ and MUC1 ＊ Between extracellular domains or between NME7-X1 and MUC1 ＊ The antibody according to claim 1, which disrupts the interaction between extracellular domains.

10. NME7 ＡＢ The antibody according to claim 1, which disrupts the binding between and PSMGFR or between NME7-X1 and PSMGFR.

11. NME7 ＡＢ The antibody according to claim 1, which disrupts the binding between N-10 and N-10 or between NME7-X1 and N-10.

12. NME7 ＡＢ and MUC1 ＊ extracellular domains or between NME7-X1 and MUC1 ＊ extracellular domains, which is the antibody according to claim 1, wherein said NME7 ＡＢ or NME7-X1 binds to said N-10 peptide (SEQ ID NO: 170) but does not bind to the C-10 peptide (SEQ ID NO: 171).

13. The antibody according to claim 2, wherein the antibody is 5A1, 4A3, 4P3, or 5D4.

14. The amino acid sequence in the heavy chain variable region, YTFTNYGMN (SEQ ID NO: 439) in the CDR1 region, WINTYTGEPTYVDDFKG (SEQ ID NO: 440) in the CDR2 region, and The amino acid sequence containing LRGIRPGPLAY (SEQ ID NO: 441) in the CDR3 region, and, The amino acid sequence in the light chain variable region, SASSSVSSYMN (SEQ ID NO: 444) in the CDR1 region, GISNLAS (SEQ ID NO: 445) in the CDR2 region, and Amino acid sequence containing QQRSSYPPT (SEQ ID NO: 446) in the CDR3 region The antibody according to claim 2, comprising:

15. The amino acid sequence in the heavy chain variable region, NTFTEYTMH (SEQ ID NO: 388) in the CDR1 region, GFNPNNNGVTNYNQKFKG (SEQ ID NO: 389) in the CDR2 region, and The amino acid sequence containing RYYHSLYVFYFDY (SEQ ID NO: 390) in the CDR3 region, and The amino acid sequence in the light chain variable region, SASQGISNYLN (SEQ ID NO: 393) in the CDR1 region, YTSSLHS (SEQ ID NO: 394) in the CDR2 region, and Amino acid sequence containing QQYSKLPYT (SEQ ID NO: 395) in the CDR3 region The antibody according to claim 2, comprising:

16. The amino acid sequence in the heavy chain variable region, NTFTEYTMH (SEQ ID NO: 429) in the CDR1 region GFNPNNNGVTNYNQKFKG (SEQ ID NO: 430) in the CDR2 region, and The amino acid sequence containing RYYHSTYVFYFDS (SEQ ID NO: 431) in the CDR3 region, and The amino acid sequence in the light chain variable region, SASQGISNYLN (SEQ ID NO: 434) in the CDR1 region, YTSSLHS (SEQ ID NO: 435) in the CDR2 region, and Amino acid sequence containing QQYSKLPYT (SEQ ID NO: 436) in the CDR3 region The antibody according to claim 2, comprising:

17. The amino acid sequence in the heavy chain variable region, NTFTEYTMH (SEQ ID NO: 388) in the CDR1 region, GFNPNNNGVTNYNQKFKG (SEQ ID NO: 389) in the CDR2 region, and The amino acid sequence containing RYYHSLYVFYFDY (SEQ ID NO: 390) in the CDR3 region, and The amino acid sequence in the light chain variable region, ITSTDIDDDMN (sequence number) in the CDR1 region, EGNTLRP (SEQ ID NO:), in the CDR2 region, and Amino acid sequence containing LQSDNLPLT (SEQ ID NO) in the CDR3 region The antibody according to claim 2, comprising:

18. The antibody according to claim 1, which is a human antibody, a humanized antibody, or a modified antibody mimetic.

19. The antibody according to claim 1, which is non-human.

20. The antibody according to claim 19, wherein the antibody is a mouse or a camel.

21. A method for administering to a patient for the prevention or treatment of cancer, comprising the step of administering to the patient a composition comprising the antibodies described in claims 1 to 17.

22. A method for preventing or treating cancer metastasis in a patient, comprising the step of administering to the patient a composition comprising the antibodies described in claims 1 to 17.

23. A method for diagnosing cancer or cancer metastasis, comprising the steps of contacting a patient sample and a normal sample with an antibody according to claims 1 to 17, and comparing the results obtained from both samples, wherein the presence of positive binding to the antibody in the patient sample indicates the presence of cancer or cancer metastasis in the patient.

24. The method according to claim 23, wherein the antibody is as defined in claim 12.

25. The method according to claim 22, wherein the antibody is linked to an imaging agent.

26. The method according to claim 22, wherein the patient specimen is blood, body fluid, tissue, or circulating cells, including during surgery, in vitro, or in vivo.

27. anti-NME7 ＡＢ Cells that have been manipulated to express antibodies or fragments thereof.

28. The cell according to claim 27, wherein the cell is an immune cell.

29. The cell according to claim 27, wherein the immune cell is a T cell or an NK cell.

30. The cell according to claim 27, wherein the cell is a stem cell or a progenitor cell.

31. The cell according to claim 30, wherein the stem cell or progenitor cell subsequently differentiates into a T cell.

32. The cell according to claim 27, comprising a chimeric antigen receptor (CAR) that recognizes a tumor-associated antigen.

33. The cell according to claim 27, wherein the expression of the anti-NME7 antibody is inducible.

34. anti-NME7 ＡＢ The cell according to claim 27, wherein a nucleic acid encoding an antibody is inserted into a Foxp3 enhancer or promoter.

35. anti-NME7 ＡＢ The cell according to claim 27, wherein the antibody is present in the NFAT induction system.

36. The cell according to claim 35, wherein the NFATc1 response element is inserted upstream of an antibody sequence that is inserted into a Foxp3 enhancer or promoter region.

37. The aforementioned anti-NME7 ＡＢ The antibody or its fragment binds to the NME7 B3 peptide, or to MUC1 ＊ NME7 to the extracellular domain of the PSMGFR peptide ＡＢ Alternatively, the cell according to claim 27, wherein the binding of NME7-X1 is disrupted.

38. The immune cell according to claim 28, which expresses a CAR that recognizes tumor-associated antigens and anti-NME7 antibodies.

39. The tumor-associated antigen is MUC1 ＊ The immune cell according to claim 38.

40. The immunogenicity-inducing portion is listed in Figures 6 to 9 as NME7. ＡＢ An anti-cancer vaccine comprising a composition containing one or more peptides derived from or peptides having at least 80%, 85%, 90%, 95%, or 97% sequence identity with respect to them.

41. The anti-cancer vaccine according to claim 40, wherein the peptide is the peptides of Sequence ID No. 141 to 145 or peptides having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

42. The anti-cancer vaccine according to claim 41, wherein the peptide is the peptide of Sequence ID No. 145 or a peptide having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

43. A BiTE comprising the antibody according to claims 1 to 17.

44. Anti-NME7 is a peptide in which the cysteine ​​residue in NME7 B3 peptide is mutated to avoid disulfide bonds. ＡＢ Methods for producing antibodies.

45. The method according to claim 44, wherein the cysteine ​​that is mutated to avoid the disulfide bond is cysteine ​​14 of the peptide of SEQ ID NO:

169.

46. Cells NME7 ＡＢ A method for generating cells with enhanced metastatic potential, comprising the step of culturing them together with NME7-X1.

47. NME7 ＡＢ Alternatively, cells that have been engineered to express NME7-X1.

48. NME7 ＡＢ Alternatively, a transgenic animal expressing NME7-X1.

49. The aforementioned NME7 ＡＢ Alternatively, NME7-X1 is human NME7 ＡＢ The transgenic animal according to claim 48, or NME7-X1.

50. The aforementioned NME7 ＡＢ Alternatively, the transgenic animal according to claim 49, wherein the expression of NME7-X1 is inducible.

51. A bispecific antibody comprising a fragment of the antibody sequence described in claims 1 to 17.

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