Bacterial NME7 orthologs and uses thereof

Abstract

The present disclosure provides for bacterial NME7 vaccines, anti-NME7 antibodies, as well as method of using such vaccines, antibodies, or variants thereof.

Description

WSGR Docket No.56699-755.601 BACTERIAL NME7 ORTHOLOGS AND USES THEREOF CROSS-REFERENCE

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 579,494 filed August 29, 2023, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on August 26, 2024, is named 56699-755_601_SL.xml and is 499,278 bytes in size. SUMMARY OF THE INVENTION

[0003] This invention is based upon the identification of NME7 orthologs in bacteria (bNME7s) having homology to human NME7. Since human NME7 promotes cancer growth and metastasis by binding to and activating the MUC1* growth factor receptor, the existence of bNME7s suggested that bNME7s may have a similar function, which could provide a mechanistic link explaining the association of certain bacteria with cancer. NME7 can activate the MUC1* receptor on cancer cells to promote growth and metastasis. Certain bacteria implicated in cancers express NME7 orthologs (bacterial NME7s or bNME7s) that can also activate MUC1*, suggesting that MUC1*-associated cancers might be prevented or treated by targeting the bNME7 – MUC1* interaction. bNME7s could be inactivated with therapeutic anti- bNME7 antibodies or with a vaccine comprising bNME7 peptides that induce an anti-bNME7 immune response. A diagnostic immunoassay to detect bNME7s in samples isolated from individuals at risk of or diagnosed with a bNME7 mediated infection or cancer could be used to identify individuals that might benefit from a vaccine or therapy targeting a bNME7.

[0004] In one aspect, the invention is an anti-cancer vaccine comprising peptides derived from human NME7 or bacterial NMEs, or consensus sequences thereof. In another aspect of the invention the anti-cancer vaccine comprises immunogenic peptides able to produce antibodies that bind to bNME7s. In another aspect, they produce antibodies that bind to bNME7s or human NME7. In another aspect, they produce antibodies that bind to bNME7s but not human NME7. In yet another aspect of the invention, are monoclonal antibodies that bind to bNME7s.

[0005] In one aspect, the invention is an NME7 vaccine. In some embodiments the vaccine comprises a peptide comprising: a sequence of SEQ ID NO: 442 or a fragment and / or variant thereof, a sequence of SEQ ID NO: 443 or a fragment and / or variant thereof, a sequence of SEQ ID NO: 444 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180,WSGR Docket No.56699-755.601 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof, or a sequence selected from SEQ ID NO 448. In some embodiments, the peptide comprises at least 8 amino acids. In some embodiments, the peptide comprises at least 10 amino acids. In some embodiments, the peptide comprises at least 12 amino acids. In some embodiments, the peptide comprises no more than 50 amino acids. In some embodiments, the peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids. In some embodiments, the variant comprises a substitution mutation. In some embodiments, the variant comprises two or three substitution mutations. In some embodiments, the peptide has higher sequence identity to a peptide of human NME7 than to any peptide of human NME1, or the peptide has less than 40% sequence identity to any peptide of human NME1. In some embodiments, the NME7 vaccine further comprised a second NME7 peptide. In some embodiments, the second NME7 peptide comprises SEQ ID NO: 81 or a fragment and / or variant thereof, a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, a sequenceWSGR Docket No.56699-755.601 selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof, or a sequence selected from SEQ ID NO 448. In some embodiments, the second NME7 peptide comprises at least 8, 10, or 12 amino acids, the second NME7 peptide comprises no more than 50 amino acids, the second NME7 peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids, the variant comprises a substitution mutation, and / or the variant comprises two or three substitution mutations. In some embodiments, the vaccine further comprises a carrier protein, adjuvant, or immunogenic agent. In some embodiments, the peptide is coupled to the carrier protein, adjuvant or immunogenic agent. In some embodiments, the peptide is connected to another peptide sequence via a spacer or linker. In one aspect, the invention is a pharmaceutical composition comprising the vaccine – e.g. the peptide. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the NME7 vaccine is formulated in a dosage unit form. In some embodiments, the vaccine is an anti-cancer vaccine.

[0006] In one aspect, the invention is a nucleic acid encoding the peptide or fragment and / or variant thereof of the vaccine above. In some embodiments, the nucleic acid is operatively linked to an expression control sequence. In some embodiments, the invention is a vector comprising the nucleic acid operatively linked to an expression control sequence. In some embodiments, the invention is a host cell comprising the vector.

[0007] In one aspect, the invention is method of isolating an anti-NME7 antibody comprising selecting an antibody that binds to an epitope comprising SEQ ID NO: 442, SEQ ID NO: 443, orWSGR Docket No.56699-755.601 SEQ ID NO: 444, or an antigenic peptide comprising a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof, or a sequence selected from SEQ ID NO 448. In some embodiments, the antigenic peptide comprises at least 8, 10, or 12 amino acids. In some embodiments, the antigenic peptide comprises no more than 50 amino acids. In some embodiments, the antigenic peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids. In some embodiments, the variant comprises a substitution mutation. In some embodiments, the variant comprises two or three substitution mutations. In some embodiments, the method further comprises injecting an animal with the peptide or an NME7 polypeptide comprising the peptide. In some embodiments, the method further comprises determining that the anti-NME7 antibody binds to an NME7 with higher affinity than it binds to NME1. In some embodiments, the method further comprises determining that the anti-NME7 antibody binds to a bacterial NME7 with higher affinity than it binds to human NME7. In some embodiments, the method further comprises determining that the anti-NME7 antibody inhibits the binding of an NME7 to MUC1*.WSGR Docket No.56699-755.601

[0008] In one aspect, the invention is an antibody that binds to an NME7 polypeptide comprising: a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof, or a sequence selected from SEQ ID NO 448. In some embodiments, the antibody binds to a human or bacterial NME7 with higher affinity than it binds to human NME1. In some embodiments, the antibody binds to a bacterial NME7 with higher affinity than it binds to human NME7. In some embodiments, the anti-NME7 antibody comprises three heavy chain (HC) complementarity determining regions (CDR) and three light chain (LC) CDRs, wherein: HC-CDR1 comprises SEQ ID NO: 41, HC-CDR2 comprises SEQ ID NO: 42, HC-CDR3 comprises SEQ ID NO: 43, LC-CDR1 comprises SEQ ID NO: 44, LC-CDR2 comprises SEQ ID NO: 45, and LC-CDR3 comprises SEQ ID NO: 46; HC-CDR1 comprises SEQ ID NO: 47, HC- CDR2 comprises SEQ ID NO: 48, HC-CDR3 comprises SEQ ID NO: 49, LC-CDR1 comprises SEQ ID NO: 50, LC-CDR2 comprises SEQ ID NO: 51, and LC-CDR3 comprises SEQ ID NO: 52; HC-CDR1 comprises SEQ ID NO: 53, HC-CDR2 comprises SEQ ID NO: 54, HC-CDR3 comprises SEQ ID NO: 55, LC-CDR1 comprises SEQ ID NO: 56, LC-CDR2 comprises SEQ IDWSGR Docket No.56699-755.601 NO: 57, and LC-CDR3 comprises SEQ ID NO: 58; HC-CDR1 comprises SEQ ID NO: 59, HC- CDR2 comprises SEQ ID NO: 60, HC-CDR3 comprises SEQ ID NO: 61, LC-CDR1 comprises SEQ ID NO: 62, LC-CDR2 comprises SEQ ID NO: 63, and LC-CDR3 comprises SEQ ID NO: 64; or HC-CDR1 comprises SEQ ID NO: 65, HC-CDR2 comprises SEQ ID NO: 66, HC-CDR3 comprises SEQ ID NO: 67, LC-CDR1 comprises SEQ ID NO: 68, LC-CDR2 comprises SEQ ID NO: 69, and LC-CDR3 comprises SEQ ID NO: 70. In some embodiments, the antibody is humanized. In some embodiments, the antibody inhibits the binding of an NME7 to MUC1*. In one aspect, the invention is pharmaceutical composition comprising the antibody. In one aspect, the invention is nucleic acid encoding the antibody. In some embodiments, the invention is an expression vector comprising a promoter and the nucleic acid. In some embodiments, the invention is a host cell comprising the expression vector.

[0009] In one aspect, the invention is diagnostic assay for detecting a bacterial NME7 ortholog (bNME7) comprising contacting a sample with any of the antibodies described above. In some embodiments, the diagnostic assay further comprises contacting the sample with a second anti- NME7 antibody. In some embodiments, the second anti-NME7 antibody binds to an epitope in the B1 region of the bNME7. In some embodiments, the second antibody is a polyclonal antibody. In some embodiments, the sample is a blood sample, a plasma sample, or a serum sample. In some embodiments, the sample is a tumor biopsy sample.

[0010] In one aspect, the invention is method of treating a MUC1* positive cancer in a subject comprising administering to the subject a pharmaceutical composition comprising an anti- NME7 antibody as described above.

[0011] In one aspect, the invention is method of preventing or treating a MUC1* positive cancer in a subject comprising administering to a subject an anti-NME7 vaccine as described above or a nucleic acid encoding an NME7 peptide of the vaccine. In some embodiments, the method inhibits metastasis of the cancer.

[0012] In one aspect, the invention is a method of preventing a MUC1* positive cancer in a subject comprising determining that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7, and administering a vaccine or nucleic acid described above to the subject. In some embodiments, the diagnostic assay described above is used to determine that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7. In some embodiments, DNA sequencing is used to determine that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7.WSGR Docket No.56699-755.601

[0013] In one aspect, the invention is a method of reducing the risk of a MUC1* positive cancer in a subject comprising: determining that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7, and administering an antimicrobial agent to the subject; thereby killing the bacteria. In some embodiments, an antimicrobial agent known to kill the bacteria is identified using the Sanford Guide.

[0014] In one aspect, the invention is a method of determining that a subject is at increased risk of a MUC1* positive cancer comprising identifying a microorganism that expresses an NME7 polypeptide in a sample from the subject, wherein the NME7 polypeptide comprises a sequence with at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to human NME7 or the B3 peptide of human NME7. In some embodiments, the method further comprises determining if the bacterial NME7 binds to the extracellular domain of MUC1*, determining if the bacterial NME7 stimulates stem cell growth or maintains stem cell pluripotency, and / or determining that the bacterial NME7 forms a dimer. BACKGROUND

[0015] There has recently been an increase in the scientific literature focused on associations between some bacteria and cancers (Zella & Gallo, 2021). Despite an increasing number of bacteria that have been linked to cancer, Helicobacter pylori is the only bacteria with clear epidemiological evidence of a causal link to cancer. A further complication is that research indicates that there are bacteria that are good for your health and bacteria that are bad, which could cause or accelerate cancer (Dzutsev et al., 2015; Lynch & Pedersen, 2016; Reid, 2018; Venkova et al., 2018; Massari et al., 2019). Some studies have shown that treatment with antibiotics has reduced tumors in animals (Zackular et al., 2015). Yet molecular mechanisms by which bacteria cause cell proliferation, self-replication, invasion or metastasis, which are the hallmarks of cancer, are still largely unknown.

[0016] Here we propose and show evidence for a mechanism of action by which certain bacteria can cause or accelerate cancers.

[0017] MUC1 is aberrantly expressed on over 75% of solid tumor cancers (Kufe et al., 1984; Gendler et al., 1990). Originally, aberrant expression meant that on healthy epithelial tissue, MUC1 was clustered at the apical border, while on cancer cells it was distributed over the entire cell surface. The inventors discovered that on cancer cells, MUC1 is cleaved by specific tumor associated enzymes, such as MMP9. Cleavage releases the entire tandem repeat domain and leaves a transmembrane protein with a truncated extra cellular domain represented by the PSMGFR peptide (Mahanta et al., 2008). Here, we refer to the MUC1 cleavage product asWSGR Docket No.56699-755.601 MUC1* (muk 1 star). Unlike full-length MUC1, MUC1* functions as a Class I growth factor receptor that activates growth and survival pathways when a ligand dimerizes its truncated extra cellular domain. We identified NME1 as a ligand that, when a dimer, binds to and dimerizes the MUC1* extra cellular domain. MUC1* dimerization induces growth of both human pluripotent stem cells and cancer cells (Hikita et al., 2008; Mahanta et al., 2008). However, at higher concentrations, NME1 forms hexamers that do not bind to and activate MUC1*. Instead, they trigger stem cell differentiation (Smagghe et al., 2013). So, NME1 limits self-replication of pluripotent stem cells by switching from the active dimer to a hexamer that induces maturation and stops self-replication. NME1 is secreted by both pluripotent stem cells and cancer cells. The paradox was how do stem cells limit self-replication but cancer cells do not. The answer was that the growth of the earliest naïve state stem cells is driven by a primitive form of NME, NME7-AB, that essentially looks like a single chain dimer of NME1, comprising an A domain, homologous to NME1, and a B domain with significant sequence variation from NME1. Secreted NME7-AB is devoid of the DM10 leader sequence and does not form higher order multimers like hexamers that could shut off self-replication. Instead, early in embryogenesis, NME7 is turned off and NME1 is turned on (Carter et al., 2016). Recently, researchers have found correlations between some bacteria and cancer or cancer metastasis. However, no cause and effect has been proposed or demonstrated. Instead, bacteria that induced cancer metastasis have been identified through a brute force, hit or miss, approach.

[0018] What is needed is a clear demonstration of a bacterium inducing cancer or cancer metastasis with an associated mechanism of action, a method of identifying bacteria that could increase cancers and a therapeutic approach for inhibiting or preventing bacterially mediated cancers.

[0019] Non-metastatic expressed 7 (NME7) functions as a growth factor that binds to MUC1*, a receptor expressed on the surface of stem cells and cancer cells. MUC1* is a truncated form of Mucin 1 (MUC1) that is generated by proteolytic cleavage. In stem cells, MUC1* activation promotes growth and maintains pluripotency. In cancer cells, MUC1* activation promotes growth and metastasis. MUC1* is expressed in 75% of all human solid tumor cancers, including breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer and esophageal cancer. MUC1* positive cancers can be treated with CAR T cells, antibody drug conjugates, or bispecific antibodies that target MUC1*, as described in WO / 2019 / 165421 and WO / 2022 / 027039 (WO / 2019 / 165421, WO / 2022 / 027039, WO / 2015 / 157322, WO / 2015 / 023694, WO / 2020 / 163325, and WO2023201234 are incorporated by reference in their entirety).WSGR Docket No.56699-755.601

[0020] NME7 comprises two nucleotide diphosphate kinase (NDPK) domains, A and B, and an N-terminal DM10 domain. The B domain lacks catalytic activity. NME7-AB and NME7-X1 are secreted and alternatively spliced forms of NME7, respectively, comprising the A and B domains, but not the DM10 domain. Binding of a single NME7 ligand to two MUC1* receptors assembles MUC1* dimers to activate intracellular signaling pathways.

[0021] NME1 is homologous to NME7, especially the A domain, but has only one NDPK domain. NME1 exists as monomers, dimers, and higher-order multimers such as hexamers. NME1 dimers activate MUC1*, whereas NME1 monomers and hexamers inhibit MUC1* (Smagghe et al., 2013).

[0022] Peptides derived from the NDPK domains of human NME7 (hNME7) were synthesized in a campaign to generate NME7 antibodies that would not cross-react with NME1, as NME1 is expressed in healthy tissues. WO / 2015 / 157322. These peptides, especially “B3”, inhibited the binding of hNME7-AB to MUC1*. Anti-B3 antibodies inhibit NME7 binding to MUC1*, NME7-mediated transformation of cancer cells to metastatic cancer stem cells, “CSCs”, and metastasis of MUC1* positive tumors, as shown in WO / 2015 / 157322, WO / 2015 / 023694 and WO / 2020 / 163325. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows a photograph of an SDS-PAGE gel onto which a recombinant Halomonas sp.593 (Hsp593) NME ortholog was run. The recombinant Hsp593 was first purified over an NTA-Ni column that captured a (His)6affinity tag, followed by separation by size exclusion chromatography using a superdex S200 column to isolate dimers. The lanes represent peak fractions eluted from the superdex S200 column.

[0024] Figure 2 is a graph of an ELISA assay showing the binding of Hsp593 NME to the extracellular domain of MUC1*. A MUC1* extra cellular domain peptide (PSMGFR) with an additional C-terminal cysteine residue was coupled to bovine serum albumin (BSA) using maleimide chemistry. Plastic surfaces in a multi-well dish were coated with the MUC1* extra cellular domain peptide / BSA conjugate and then incubated with His-tagged Hsp593 NME. Bound bNME7 was detected using a horse radish peroxidase (HRP) coupled anti-His antibody.

[0025] Figure 3 shows a graph of the expression of metastatic markers and stem cell pluripotency genes as measured by RT-PCR. Human T47D breast cancer cells were grown for 10 days in a minimal, serum-free media containing an Hsp593 NME7 as the only potential growth factor. After 10 days, “Floater” cells which became non-adherent were collected and analyzed by RT-PCR. The PCR measurement is a fold-increase over the control, T47D cells grown in standard, serum containing RPMI media.WSGR Docket No.56699-755.601

[0026] Figures 4A-4B show an amino acid sequence alignment of cancer associated bNME7 polypeptides identified by searching for orthologs of the B domain of human NME7. A GTIR motif (SEQ ID NO: 448) that distinguishes many bNME7 sequences from human NME7 is underlined along with a core portion of the B3 peptide.

[0027] Figure 5 shows an alignment of the CanBac1, CanBac2, and CanBac3 bacterial NME7 consensus sequences that were used to identify additional bNMEs.

[0028] Figures 6A-6B show amino acid sequence alignments of the CanBac3 sequence to human NME1 (Fig.6A) and the A domain of human NME7 (Fig.6B).

[0029] Figures 7A-7B show an amino acid sequence alignment of cancer associated bNME7 polypeptides identified by searching for orthologs of the CanBac3 peptide. A GTIR motif (SEQ ID NO: 448) that distinguishes many bNME7 sequences from human NME7 is underlined along with a conserved sequence in a region of the bNME7s that aligns to a sequence within the B3 peptide of human NME7a.

[0030] Figures 8A-8B show an amino acid sequence alignment of cancer associated bNME7 polypeptides identified by searching for orthologs of the CanBac2 peptide. A GTIR motif (SEQ ID NO: 448) that distinguishes many bNME7 sequences from human NME7 is underlined along with a conserved sequence in a region of the bNME7s that aligns to a sequence within the B3 peptide of human NME7a.

[0031] Figures 9A-9B show an amino acid sequence alignment of cancer associated bNME7 polypeptides identified by searching for orthologs of the CanBac1 peptide. A GTIR motif (SEQ ID NO: 448) that distinguishes many bNME7 sequences from human NME7 is underlined along with a conserved sequence in a region of the bNME7s that aligns to a sequence within the B3 peptide of human NME7a.

[0032] Figure 10 is an overlay of traces from the analysis of a selection of newly identified cancer-associated bNME7s that were recombinantly expressed then analyzed by size exclusion chromatography using a superdex S200 column. The chromatography profiles of the bNME7s show they can form dimers, tetramers, or hexamers.

[0033] Figures 11A-11B show further demonstration of the purity of the recombinant bNME7s. Fig.11A is a Coomassie stained non-reducing SDS polyacrylamide gel demonstrating the purity of some isolated bNME7s. Fig.11B is a table listing the abbreviated and full name of each bacterium from which the bNME7s were derived.

[0034] Figure 12 is a graph of an ELISA assay where a MUC1* extra cellular domain peptide has been adsorbed to a multi-well plate. Recombinant Hsp593 NME7, or truncated A. baumanni NME7, or human NME7-AB, all bearing a histidine tag, were incubated with the MUC1*WSGR Docket No.56699-755.601 peptide presenting surface. Bound NMEs were detected using a horse radish peroxidase (HRP) coupled anti-His antibody.

[0035] Figure 13A-13B show consensus sequences for bNME7s. Fig.13A shows a consensus sequence of the B3 region of 15 bNME7s that are cancer associated and bind strongly to B3C and B3D (A415>0.5). Fig.13B shows a consensus sequence of the B3 region of 70 bNME7s identified in a BLAST search, where their association to cancer was not considered.

[0036] Figure 14 is an alignment of overlapping peptides derived from the human NME7B3 peptide that were used for epitope mapping of a panel of antibodies that bind to the human B3 peptide. Each peptide has a C-terminal Cysteine, which was added to facilitate coupling to BSA to facilitate adsorption to a surface for ELISA assays. A Cysteine within the B3 sequence is replaced by Serine so as not to interfere with coupling.

[0037] Figure 15 shows a graph from an ELISA experiment to determine to which portions of the B3 peptide various anti-NME7 antibodies bind. The peptides shown in Fig.14 were adsorbed onto a multi-well plate. Various anti-NME7 antibodies that bind to B3 peptide are added and then detected.

[0038] Figure 16 is a graph of an ELISA experiment in which the binding of various bNME7s to a polyclonal antibody that binds to the B1 peptide of human NME7 is measured.

[0039] Figures 17A – 17C show photographs of Western blots from a co- immunoprecipitation experiment performed on human stem cells. An antibody against the MUC1 cytoplasmic tail (Ab-5) was used to immune-precipitate MUC1* from extracts of human induced pluripotent stem cells (iPS7) or embryonic stem cells (HES3) and compared to a control precipitation with a non-specific antibody (IgG). The immunoprecipitated proteins were then run on an SDS-PAGE gel and transferred to a membrane. Fig.17A shows a Western blot probed with B9, a commercially available anti-NME7 antibody. NME7 polypeptides migrating at ~33kDa and ~30kDa were detected in both MUC1* immune-precipitates, whereas a larger (full- length) NME7 species of ~42 kDa was detected in the lysate. Fig.17B shows the same gel after it was stripped and re-probed with an antibody against the extracellular domain of MUC1* (anti- PSMGFR), confirming that MUC1* was precipitated. Fig.17C shows a Western blot of a mixture of recombinant hNME7-AB and recombinant hNME7-X1 probed with the anti-NME7 antibody. This experiment demonstrates that an NME7-AB-like species and NME7-X1 are naturally occurring in stem cells and interact with MUC1*.

[0040] Figures 18A-18E shows photographs of Western blots of a co-immunoprecipitation experiment performed on human T47D breast cancer cells. An antibody against the MUC1 cytoplasmic tail (Ab-5) was used to immune-precipitate MUC1* from extracts of human T47D breast cancer cells and compared to a control precipitation with a non-specific antibody (IgG).WSGR Docket No.56699-755.601 The immunoprecipitated proteins were then run on an SDS-PAGE gel and transferred to a membrane. Fig.18A shows a Western blot probed with B9, a commercially available anti- NME7 antibody. NME7 polypeptides migrating at ~33kDa and ~30kDa. Fig.18B shows a Western blot probed with 3F7, a different commercially available anti-NME7 antibody. The arrows indicate that both gels show unique NME7 bands at ~33kDa and ~30kDa. We note that the NME7 species immunoprecipitated from the cell lysate runs with an apparent molecular weight of 42 kDa which is the weight of full-length hNME7. Fig.18C shows a recombinant hNME7-AB and a recombinant hNME7-X1 that we made were mixed together and run on a gel, then probed with an anti-NME7 antibody, showing that the two unique NME7 species, ~33kDa and ~30kDa, that are naturally occurring in breast cancer cells are NME7-AB and NME7-X1. Fig.18D shows the gel above was stripped and re-probed with an antibody against the extracellular domain of MUC1*, anti-PSMGFR. Fig.18E) shows the gel above was stripped and re-probed with an antibody against the extracellular domain of MUC1*, anti-PSMGFR.

[0041] Figure 19A-19T show photographs of immunofluorescent detection of human NME7 in a wide range of different cancer subtypes, wherein all are MUC1* positive. Cells were probed with a fluorescently labeled anti-NME7 antibody that binds to the B3 peptide, called 5A1, at concentrations ranging from 0.5 ug / mL to 8 ug / mL. Figs.19A-19D show T47D breast cancer cells probed with anti-NME7 antibody 5A1. Figs.19E-19H show ZR75-1 breast cancer cells probed with the same anti-NME7 antibody. Figs.19I-19L show H1975 non-small cell lung cancer cells probed with the same anti-NME7 antibody. Figs.19M-19P show HPAFII pancreatic cancer cells probed with the same anti-NME7 antibody. Figs.19Q-19T show DU145 prostate cancer probed with the same anti-NME7 antibody.

[0042] Figure 20A-20B shows two tables showing that the generation of floating cells, presumably cancer stem cells, is nearly eliminated by adding anti-NME7 antibodies. The addition of antibody #61 that binds to the B3 domain and blocks the interaction with MUC1* essentially blocked the generation of the floater cells. Because the table contains scientist observations, two researchers independently viewed the resultant cells and independently estimated the percentage of the cells that were floating. Fig.20A shows the observations of PhD scientist JR. Fig.20B shows the observations of VH.

[0043] Figure 21 is a table showing the amount of total RNA produced when human T47D breast cancer cells are cultured for 10 days in either standard serum containing media or in a minimal media containing either two biochemical inhibitors (2i) or hNME7-AB. To determine which effects were mediated by NME7, an anti-NME7 antibody that binds to the B3 peptide was added. As can be seen in the table, the addition of the anti-NME7 antibody blocked the growth of the cancer cells.WSGR Docket No.56699-755.601

[0044] Figure 22 shows a graph of the expression of CXCR4, measured in a PCR assay, for each condition. Note that even though we have not added exogenous NME7 to the 2i condition, the addition of the anti-hNME7 B3 antibody inhibited the upregulation of CXCR4. The arrows point from T47D cancer cell grown in 2i minimal media to 2i minimal media plus anti-hNME7 antibody; the fold increase in CXCR4 expression, compared to growth in RPMI media falls from a 10-fold increase to zero in the presence of the antibody. The second arrow points from the cancer cells grown in hNME7-AB, where CXCR4 is expressed 70-fold higher than the control grown in RPMI media, to where CXCR4 fold increase in expression drops to only 10-fold. hNME7-AB or 2i in Minimal Media upregulate CXCR4, a metastatic marker for breast cancers. Addition of an anti-NME7 antibody abolishes or greatly decreases CXCR4 expression.

[0045] Figure 23A-23B shows graphs of PCR measurements after human T47D breast cancer cells are cultured in minimal media alone for 10 days. Fig.23A is a graph of a PCR experiment in which T47D breast cancer cells that were cultured for 10 days in minimal media alone have increased expression of hNME7 and the alternative splice isoform hNME7-X1. Fig.23B is a graph of a PCR experiment in which T47D cells cultured in minimal media alone have increased expression of MUC1.

[0046] Figure 24A-24D shows PCR graphs of measurements of metastatic marker and stem cell pluripotency genes after human T47D breast cancer cells were cultured for 10 days in minimal media plus various bNME7s that showed high sequence homology to the B3 peptide region of hNME7. Fig.24A is a graph of PCR measurement of breast cancer metastatic marker CXCR4 in cancer cells grown for 10 days in minimal media plus the bNME7s indicated, in the cells that had become non-adherent, “Floaters” as well as the adherent cells. Fig.24B shows measurement of pluripotency gene SOX2. Fig.24C shows measurement of pluripotency gene OCT4. Fig.24D shows measurement of pluripotency gene NANOG. As can be seen, metastatic marker CXCR4 and pluripotency markers SOX2, OCT4 and NANOG have increased expression, especially in the floater population, when cultured in bNME7s.

[0047] Figure 25A-25C shows the characteristics of human stem cells that were cultured in minimal media plus a bNME7. The bacterial NME7s induced robust stem cell growth and pluripotency as well as the naïve state over a 144-hour period. Fig.25A is a graph measuring the percent confluency of the stem cells, as an indicator of growth induced by the bNME7s. Percent confluence was measured digitally using Image J on photographs of the stem cells 96 hours post addition of the bNME7s. Fig.25B is a graph of OCT4 positivity, indicating the pluripotency of the stem cell population, also measured 96 hours after growth in bNME7 was initiated. OCT4 was measured by digital image analysis of integrated pixel density of stem cells stained with an OCT4 antibody bearing a green fluorescent tag. Fig.25C is a graph of H3K27me, indicating theWSGR Docket No.56699-755.601 X chromosome activation status. H3K27me was measured by digital image analysis of integrated pixel density of stem cells stained with an H3K27me antibody bearing a red fluorescent tag. Anti-H3K27me is used to indicate the activation status of the X chromosomes in female source stem cells.

[0048] Figures 26A-26R show photographs of stem cells at various stages of pluripotency as indicated by OCT4 positivity and X chromosome activation status as measured by an H2K27me antibody. Figs.26A-26G show photographs of stem cells stained for the expression of OCT4 and a scoring system from 1 to 4 for assessing pluripotency, where 1 is the highest and 4 is the lowest. The portions indicated by dotted line indicates areas of cells where OCT4 intensity is reduced by 50%, 75% or 100% compared to the typical bright OCT4 staining. Figs.26I-26P show photographs of stem cells stained for the expression of H3K27me that indicates that both X chromosomes are active or where one X has been inactivated. The photographs and corresponding scoring system from 1 to 4 for assessing XX activation, where 1 is the highest and 4 is the lowest. Fig.26Q shows a graph of the percentages of stem cells that are 100% pluripotent and OCT4 positive, or at various stages of differentiation as detailed in the scoring scheme above the graph. Fig.26R shows a graph of the percentages of naïve state stem cells that are 100% XaXa, meaning both X chromosomes are still active or at various stages of differentiation toward inactivating one X, as detailed in the scoring scheme above the graph.

[0049] Figure 27 shows is a table summarizing the ability of the bNME7s to promote stem cell growth and their corresponding binding to MUC1* relative to that of hNME7-AB. The table also shows their multimerization state as determined by size exclusion chromatography. Here we note that the bNME7s that resulted in the highest rates of stem cell growth were dimers.

[0050] Figure 28 shows the table of dimers only, reflecting a near perfect correlation between strength of binding to MUC1* extra cellular domain peptide and growth rate of stem cells cultured in that bNME7.

[0051] Figures 29A-29W show bioluminescent photographs from an IVIS instrument demonstrating treatment of metastatic breast cancer with anti-NME7 antibodies. The tails veins of female nu / nu mice were injected with T47D breast cancer cells expressing a luciferase tracer. The T47D cells were either cultured in regular RPMI media (T47D WT) or cultured for 10 days in minimal media supplemented with hNME7-AB (T47D CSC).500,000 of the T47D WT cells were injected versus only 10,000 of the T47D CSCs. The T47D WT cells barely grew in the mice (Fig.29A) whereas the T47D CSCs metastasized by Day 10 (Fig.29B). A second mouse that had been injected with T47D CSCs was treated with 15 mgs / kg of a cocktail of anti-NME7 monoclonal antibodies 5A1, 4A3, and 5D4 that bind to the NME7 B3 peptide. The metastases observed in this mouse at Day 10 (Fig.29C) started to regress by Day 12 (Fig.29D) and wereWSGR Docket No.56699-755.601 almost completely gone by Day 14 (Fig.29E). Figs.29F-29N show a time-series of photographs of the mouse injected with T47D CSCs and then treated with anti-NME7 antibodies. Metastases reappeared after treatment was stopped at Day17 and then regressed again after the anti-NME7 antibody treatment resumed at Day 21. Figs.29O-29W show a time- series of photographs of the mouse injected with T47D WT cells. T47D WT cancer cells were abundant on Day 6 shortly after the injection of 500,000 cells, but did not sustain metastases, even in the absence of treatment, leaving the mouse cancer free at the end of the experiment on Day 31.

[0052] Figure 30A-30G show bioluminescent photographs of human breast cancer metastases in mice demonstrating that bNMEs can generate cancer stem cells and support their metastasis growth. T47D breast cancer cells were cultured in the indicated bNMEs.30,000 floating cells that had lost adhesion to the culture dish were intravenously injected into nu / nu mice pretreated with 90-day release estrogen pellets. Post transfer, the mice were injected twice per week with the same recombinant bNME. By Day 18, metastases had formed in mice injected with T47D CSCs activated by recombinant NME7 proteins from A. baumannii (Fig.30A), Halomonas heilongjiangensis (Fig.30B), Gammaproteobacteria bacterium (Fig.30C), human (Fig.30D), Halomonas sp.593 (Fig.30E), Gammaproteobacteria bacterium (Fig.30F) and Chlamydiae bacterium (Fig.30G). These results demonstrate that bNME7 grown cancer cells induced metastasis from injection of only 30,000 cells.

[0053] Figure 31 is a table of consensus sequence peptides that were designed for use in producing anti-cancer antibodies and incorporation into vaccines. The table lists the consensus sequence peptides and their sequence identity to human NME7 or bacterial NME7s. Here sequence identity was to the entire peptide, rather than to a portion of the peptide. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0054] In the present application, “a” and “an” are used to refer to both single and a plurality of objects.

[0055] As used herein, “about” or “substantially” generally provides a leeway from being limited to an exact number. For example, as used in the context of the length of a polypeptide sequence, “about” or “substantially” indicates that the polypeptide is not to be limited to the recited number of amino acids. A few amino acids add to or subtracted from the N-terminus or C-terminus may be included so long as the functional activity such as its binding activity is present.WSGR Docket No.56699-755.601

[0056] As used herein, administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0057] As used herein, "amino acid" and "amino acids" refer to all naturally occurring L- ^- amino acids. This definition is meant to include norleucine, ornithine, and homocysteine.

[0058] As used herein, in general, the term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a reference (e.g. native sequence) polypeptide. The amino acid alterations may be substitutions, insertions, deletions or any desired combinations of such changes in a native amino acid sequence.

[0059] Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0060] Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are proteins or fragments or derivatives thereof which exhibit the same or similar biological activity and derivatives which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and so on.

[0061] Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native amino acid sequence. Immediately adjacent to an amino acid means connected to either the ^-carboxy or ^-amino functional group of the amino acid.

[0062] Deletional variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.

[0063] As used herein, “fragments” or “functional derivatives” refers to biologically active amino acid sequence variants and fragments of the polypeptide of the present invention, as well as covalent modifications, including derivatives obtained by reaction with organic derivatizing agents, post-translational modifications, derivatives with nonproteinaceous polymers, and immunoadhesins.WSGR Docket No.56699-755.601

[0064] As used herein, "vaccines" include any preparation that is used to stimulate the body’s immune response against a disease. The vaccines can comprise a peptide, or peptides, or the nucleic acids that encode them, including in the context of a recombinant vaccine, a DNA vaccine, RNA vaccine, or mRNA vaccine, which may be in the context of a suitable carrier or adjuvant. The vaccines can be administered through needle injections, microneedle patch, by mouth or by nasal spray.

[0065] As used herein, "carriers" include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include without limitation buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; 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 dextrins; 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®.

[0066] As used herein "pharmaceutically acceptable carrier and / or diluent" includes any and all solvents, dispersion media, coatings antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0067] It is especially advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.WSGR Docket No.56699-755.601

[0068] As used herein, "vector", "polynucleotide vector", "construct" and "polynucleotide construct" are used interchangeably herein. A polynucleotide vector of this invention may be in any of several forms, including, but not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged in another viral or viral-like form (such as herpes simplex, and adeno- structures, such as polyamides.

[0069] As used herein, "host cell" includes an individual cell or cell culture which can be or has been a recipient of a vector of this invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or change.

[0070] As used herein, "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0071] As used herein, "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, and so on. Preferably, the mammal is human.

[0072] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Palliating" a disease means that the extent and / or undesirable clinical manifestations of a disease state are lessened and / or the time course of the progression is slowed or lengthened, as compared to a situation without treatment.

[0073] As used herein, “A1” peptide, “A2” peptide, “B1” peptide, “B2” peptide and “B3” peptide refer to peptides used to generate antibodies that bind to human NME7-AB, but not (or significantly less) to human NME1. The peptides used to generate these antibodies are common to both NME7-AB and NME7-X1, and are set forth as below.

[0074] A1 is NME7A peptide 1 (A domain): MLSRKEALDFHVDHQS (SEQ ID NO: 77)

[0075] A2 is NME7A peptide 2 (A domain): SGVARTDASES (SEQ ID NO: 78)

[0076] B1 is NME7B peptide 1 (B domain): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 79)

[0077] B2 is NME7B peptide 2 (B domain): EVYKGVVTEYHDMVTE (SEQ ID NO: 80)WSGR Docket No.56699-755.601

[0078] B3 is NME7B peptide 3 (B domain): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 81)

[0079] Further, for the sake of clarity, NME7A (with capital letter “A”) refers to the subunit A portion of NME7. NME7B (with capital letter “B”) refers to the subunit B portion of NME7.

[0080] As used herein, an “effective amount” of an agent to inhibit an NME7 protein refers to an amount of the agent which inhibits the activating interaction between the NME7 protein and MUC1*.

[0081] As used herein, the “extracellular domain of MUC1*” refers to the extracellular portion of a MUC1 protein that is devoid of the tandem repeat domain. In most cases, MUC1* is a cleavage product wherein the MUC1* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail. The precise location of cleavage of MUC1 is not known perhaps because it appears that it can be cleaved by more than one enzyme. The extracellular domain of MUC1* will include most of the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 3) but may have an additional 10-20 N-terminal amino acids or 10-20 deletions from the N- terminus.

[0082] As used herein, NME proteins” have at least one NDPK (nucleotide diphosphate kinase) domain. In some cases, the NDPK domain is not functional in terms of being able to catalyze the conversion of ATP to ADP.

[0083] Human NME7 can mean the native protein or a variant, such as NME7-AB that has superior commercial applicability because variations allow high yield expression of the soluble, properly folded protein in E. coli. NME7-AB consists primarily of the NME7 A and B domains but is devoid of most of the DM10 domain, which is at the N-terminus of the native protein. NME7-AB may be a cleavage product of full length NME7. Other variants include NME7-X1 and NME7-X1 like proteins whose nucleic acid sequence is devoid of all or most of a DM10 domain. Full length human NME7 as referred to herein is intended to mean native human NME7 having a molecular weight of about 42kDa.

[0084] As used herein, the term “cancer stem cells” refers to cancer cells that express levels of genes that have been linked to a more metastatic state or more aggressive cancers. The term “cancer stem cells” can also refer to cancer cells for which far fewer cells are required to give rise to a tumor when transplanted into an animal. Cancer stem cells are often resistant to chemotherapy drugs. Another term for “cancer stem cells”, CSCs, is “tumor initiating cells”.

[0085] As used herein, the terms “stem / cancer”, “cancer-like”, “stem-like” refers to a state in which cells acquire characteristics of stem cells or cancer cells, share important elements of the gene expression profile of stem cells, cancer cells or cancer stem cells. Stem-like cells may beWSGR Docket No.56699-755.601 somatic cells undergoing induction to a less mature state, such as increasing expression of pluripotency genes. Stem-like cells also refers to cells that have undergone some de- differentiation or are in a meta-stable state from which they can alter their terminal differentiation. Cancer-like cells may be cancer cells that have not yet been fully characterized but display morphology and characteristics of cancer cells, such as being able to grow anchorage-independently or being able to give rise to a tumor in an animal.

[0086] As used herein, “spacers” or “linkers” of different lengths can be incorporated anywhere in the peptide. Spacer attachment is usually through an amide linkage but other functionalities are possible. Concept

[0087] This invention is based upon the identification of NME7 orthologs in bacteria (bNME7s) having homology to human NME7. Since human NME7 promotes cancer growth and metastasis by activating MUC1*, the existence of bNME7s suggested that bNME7s may have a similar function, which could provide a mechanistic link explaining the association of certain bacteria with cancer.

[0088] Bacterial NME7 orthologs homologous to the full human NME7 sequence were used to design bacterial NME7 B3 consensus sequences, which were then used to identify additional bacterial NME7 orthologs. bNME7 derived from bacteria known to be associated with cancer were expressed recombinantly and shown to bind to PSMGFR, a peptide representing the extracellular domain of MUC1*. bNME7s that could bind PSMGFR were then shown to activate MUC1* in stem cells and cancer cells, consistent with a hypothesis that bNME7s cause cancer and cancer metastasis. Background to linking NMEs to correlation between some bacteria and cancers

[0089] To facilitate previous experiments focused on the conflicting roles of NME1 dimers versus hexamers, we needed to make recombinant NME1 that formed more stable dimers than the wild type. One way was to introduce a mutation, S120G, that had been identified in a brain cancer. Another way was to delete amino acids from the C-terminus of NME1. In our curiosity about which species express NMEs, we learned that NMEs are expressed in mammals and many other organisms including bacteria. Because bacteria don’t mature or differentiate, we wondered if their NMEs were also functioning as growth factors. In sequence alignments to human NME1 we discovered that bacteria have a truncated C-terminus, similar to the C-terminal deletions we used to make stable human NME1 dimers.WSGR Docket No.56699-755.601

[0090] One unexpected hypothesis that we proposed was that some bacterial NMEs could look so much like human NME1 or like the B3 region of human NME7, which we showed interacts with MUC1* extra cellular domain, that they could mimic human NME7 and cause or accelerate cancer cell growth or metastasis.

[0091] We had previously discovered that monomeric human NME7-AB comprising the A and B domain, but devoid of the DM10 leaders sequence: 1) bound to the MUC1* PSMGFR peptide of MUC1* extra cellular domain; 2) promoted the growth and pluripotency of human stem cells; 3) promoted the growth of human cancer cells; and 4) induced the transformation of human cancer cells to a more metastatic state, which was reversed by an anti-NME7 antibody that blocked the interaction of the B3 peptide region to the MUC1* extra cellular domain.

[0092] Here we have demonstrated that bacterial NME7s with high sequence homology to the B3 peptide region of hNME7 mimic hNME7-AB in their biological function. We showed that the bacterial NME7s : 1) bind to the PSMGFR peptide of MUC1* extra cellular domain; 2) induce human stem cell growth and pluripotency; 3) support human cancer cell growth; and 4) induce transformation of cancer cells to a more metastatic state, able to form tumors in mic from injection of a small number of cells compared to the number of parent cell required to form stable tumors. markers, such as CXCR4 in breast cancers, and stem cell markers, such as OCT4, NANOG, and SOX2 in cancer cells. Adjuvants

[0093] Adjuvants enhance the adaptive immunity of vaccines by activating innate immune cells (Zhao et al., 2023). Adjuvants can be categorized as immunostimulants and delivery systems. Immunostimulants are danger signal molecules that lead to the maturation and activation of antigen-presenting cells (APCs) by targeting Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) to promote the production of antigen signals and co- stimulatory signals, which in turn enhance the adaptive immune responses. On the other hand, delivery systems are carrier materials that facilitate antigen presentation by prolonging the bioavailability of the loaded antigens, as well as targeting antigens to lymph nodes or APCs.

[0094] Vaccines of the present invention may comprise an adjuvant. In some embodiments, the adjuvant comprises aluminum hydroxide and / or aluminum phosphate. In some embodiments, the adjuvant comprises mineral salts, microbial products, emulsions, saponins, synthetic small molecule agonists, polymers, nanoparticles, or liposomes. In some embodiments, the adjuvant comprises an oil-in-water emulsion. The oil-in-water emulsion may comprise squalene, Tween 80 and Span 85. The oil-in-water emulsion may comprise alpha-tocopherol, squalence, and Tween 80. The oil-in-water emulsion may comprise mineral oil and mannideWSGR Docket No.56699-755.601 monooleate. The oil-in-water emulsion may comprise non-mineral oil and mannide monooleate. In some embodiments, the adjuvant comprises LPS or a non-toxic LPS derivative or mimic such as monophosphoryl lipid A or Glucopyranosyl lipid A. In some embodiments, the adjuvant comprises MPLA and QS-21. In some embodiments, the adjuvant comprises detoxified LPS and alum. In some embodiments, the adjuvant comprises a nucleotide or nucleic acid, such as CpG oligodeoxynucleotides, Cyclic dinucleotides, double-stranded RNA, or single-stranded DNA. In some embodiments, the adjuvant comprises a lipid nanoparticle comprising ionizable lipids, phospholipids, cholesterol, and / or polyethylene glycol modified lipids. In some embodiments, the adjuvant comprises an imidazoquinoline. In some embodiments, the adjuvant comprises a small-molecule inhibitors targeting mevalonate pathways such as a lipophilic statin. In some embodiments, the adjuvant comprises an mTOR inhibitor. In some embodiments, the adjuvant comprises Manganese. In some embodiments, the adjuvant comprises Chitosan. In some embodiments, the adjuvant comprises a synthetic polymeric particle material such as PLGA. In some embodiments, the adjuvant comprises virus-like particles, caged protein nanoparticles, mesoporous silica nanoparticles, or gold nanoparticles. In some embodiments, the adjuvant comprises an injectable hydrogel or self-assembled scaffolds with macroporous structures for slow antigen release.

[0095] In one embodiment, a vaccine comprising a bNME7 or consensus bNME7 peptide and an adjuvant is administered to a subject with cancer. Combination therapy, cross-reactivity, and cross-protection

[0096] It is well known that vaccines can induce immune responses to antigens other than those contained in the vaccine, termed “cross-reactivity” (Vojtek et al., 2019). Vaccination using specific antigen targets can induce immune responses towards additional pathogen strains (and sometimes even different species). This forms the basis of cross- protection in vaccination, in which clinical protection beyond the target antigen(s) is observed. Ideal vaccine antigens therefore include epitopes with conserved homology across related pathogen types, because it is not always possible to include the antigens of all the individual types of a given pathogen species. A single antigen typically contains various different epitopes, which can be recognized by multiple different clones of B cells. This leads to proliferation of all the different clones and is known as a polyclonal response. An immune response to an antigen therefore classically raises many different antibodies to the various exposed epitopes on the antigen. Such polyclonal responses provide one of the main underlying principles of cross-reactivity. Furthermore, adjuvants have the benefit of potentially improving cross-reactivity. By selecting and combiningWSGR Docket No.56699-755.601 the most appropriate adjuvant components for a specific antigen, it is possible to design and formulate vaccines that deliver greater and broader protection.

[0097] Accordingly, combinations of bNME7 peptides from different bacteria and bNME7 consensus peptides can be used to elicit an immune response to a broad spectrum of bNME7s, including bNME7s that differ in sequence from the peptides in a vaccine, and even bNME7s not disclosed in this application. The Hsp593 NME ortholog induces expression of metastatic and pluripotency genes in cancer cells

[0098] Halomonas sp.593 (Hsp593) expresses an NME ortholog that forms dimers (Georgescauld et al., 2013). A double-stranded cDNA fragment encoding Hsp593 NME was inserted between the NdeI and XhoI restriction sites of pET21b. The resulting expression vector was transformed into BL21(DE3) competent E. coli. Recombinant protein was expressed in Magic Media (Thermofisher), according to the manufacturer’s instructions. The proteins were purified by a two-step purification method: 1) affinity chromatography using a NiNTA column and 2) size exclusion chromatography using a superdex S200 column to isolate dimers (Fig.1).

[0099] Hsp593 NME binding to the extracellular domain of MUC1* was demonstrated by ELISA. A MUC1* extra cellular domain peptide (PSMGFR, SEQ ID NO: 3) with an additional C-terminal cysteine residue was coupled to bovine serum albumin (BSA) using maleimide chemistry. Plastic surfaces in a multi-well dish were coated with the MUC1* ecd / BSA conjugate and then incubated with His-tagged Hsp593 NME. Bound bNME7 was detected using an HRP coupled anti-His antibody (Fig.2).

[0100] Recombinant Hsp593 NME induced expression of CXCR4, a marker of metastatic breast cancers, and pluripotency genes Oct4, Sox2, and Nanog, as determined by RT-PCR, in MUC1*-positive T47D breast cancer cells. The cells were cultured for 10 days in a serum free minimal media with recombinant Hsp593 NME or in normal RPMI growth media. ‘Floaters’ refers to cells that became non-adherent (Fig.3). As can be seen in the figure, the metastatic marker for breast cancers, CXCR4, has increased by 200-fold. Stem cell pluripotency genes NANOG, OCT4 and SOX2 have increased by 300-fold, 290-fold, and 60-fold, respectively, compared to the T47D RPMI control. We note that CDH1 which is a metastatic marker for prostate cancer has not increased.

[0101] Hsp593 is linked to gingivitis. Patients with chronic gingivitis and related conditions are at increased risk of developing cancers, including but not limited to oral cancers (Wroblewski et al., 2010; Martinez-Garcia & Hernandez-Lemus, 2021). These observations suggest that Hsp593 NME7 may contribute to cancer and / or cancer metastasis.WSGR Docket No.56699-755.601 Many microorganisms express NME7 orthologs

[0102] Helicobacter pylori, a bacterium that causes ulcers, has been associated with an increased risk of gastric cancer (Wroblewski et al., 2010). The H. pylori NME ortholog has a similar degree of homology to the NDPK B domain of human NME7 as the NME ortholog of Hsp593, as shown in Table 1. Table 1. Percent identity of B domain of human NME7 to its Hsp593, and H. pylori NME orthologs

[0103] Additional bacteria with NME7 homologs were identified in a BLAST search against the B domain of NME7, as shown in Table 2. Table 2. Top 100 bacteria with NME proteins homologous to the B domain of NME7, ranked by E-value.WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601Many bacteria associated with cancer have NME7 orthologs

[0104] Surprisingly, many bacterial species with bNME7s homologous to hNME7 are associated with cancer in the literature, as shown in Table 3. Table 3. Bacteria with NME7 orthologs that are also associated with cancer.WSGR Docket No.56699-755.601

[0105] An alignment comparing the amino acid sequences of cancer-associated bNME7 orthologs of Table 3 is presented in Figs.4A-4B. Designing search sequences to identify more bNME7s

[0106] Three search sequences were designed based upon sequences found within the B3 region of bNME7s. CanBac 1: ARFGVDKIKNAVHCTDLPEDAELEVNYFF SEQ ID NO: 83 CanBac 2: ADFGNTSIENAVHGSDSPENALLEIQYFF SEQ ID NO: 84 CanBac 3: AIFGNTKIENAVHGSDSPENALLEVQYFF SEQ ID NO: 82

[0107] The search sequences were aligned with each other to illustrate their similarities with each other and human NME7 (Fig.5).

[0108] BLAST analysis of the consensus sequences against the human genome demonstrates homology to NME7, but not to other human proteins, as shown in Table 4.WSGR Docket No.56699-755.601 Table 4.

[0109] CanBac3 has low homology to NME1 (Fig.6A) and the A domain of NME7 (Fig.6B).

[0110] Antibodies generated against these consensus sequences may bind to many bNME7s that can mimic human NME7s and activate MUC1*.

[0111] The panel of bacterial NME7 orthologs was expanded by identifying the bacterial genes most similar to each search sequences. Table 5 shows results from a BLAST search with the CanBac3 consensus sequence. Only proteins annotated as NMEs are shown. Table 5. Bacteria with NME proteins having homology to the CanBac3 bacterial consensus peptide.WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601

[0112] Many of the bNME7s identified by searching with the CanBac3 bacteria consensus peptide have been associated with cancer in the literature, as shown in Table 6. Table 6. Bacteria from Table 5 that are associated with cancer.WSGR Docket No.56699-755.601

[0113] An alignment comparing the amino acid sequences of the cancer-associated bNME7 orthologs of Table 6 is presented in Figs.7A-7B.

[0114] Table 7 shows results from a BLAST search with the CancerBac2 consensus sequence. Only proteins annotated as NMEs are shown. Table 7. Bacteria with NME proteins having homology to the CancerBac2 consensus.WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601

[0115] Many of the bNME7s identified by searching with the CanBac2 consensus peptide have been associated with cancer in the literature, as shown in Table 8. Table 8. Bacteria from Table 7 that are associated with cancer.WSGR Docket No.56699-755.601

[0116] An alignment comparing the amino acid sequences of cancer-associated bNME7 orthologs of Table 8 is presented in Figs.8A-8B.

[0117] Table 9 shows results from a BLAST search with the CanBac1 consensus sequence. Only proteins annotated as NMEs are shown. Table 9. Bacteria with NME proteins having homology to the CancerBac1 consensus sequence.WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601

[0118] Many of the bNME7s identified by searching with the CanBac1 consensus peptide are also associated with cancer in the literature, as shown in Table 10. Table 10. Bacteria from Table 9 that are associated with cancer.WSGR Docket No.56699-755.601

[0119] An alignment comparing the amino acid sequences of cancer-associated bNME7 orthologs of Table 10 is presented in Figs.9A-9B.

[0120] A summary of exemplary cancer-associated bNME7s identified thought the BLAST searches is presented in Table 11. Table 11. Cancer-associated bNME7 polypeptides.WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601WSGR Docket No.56699-755.601Recombinant bNME7 proteins assemble into dimers and higher order multimers

[0121] A selection of newly identified cancer-associated bNME7s were recombinantly expressed and isolated by 1) affinity chromatography using a NiNTA column and 2) size exclusion chromatography using a superdex S200 column. Chromatography profiles of bNME7s that form dimers, tetramers, and hexamers are shown in Fig.10. A Coomassie stained non- reducing SDS polyacrylamide gel demonstrating the purity of some isolated bNME7s is shown in Fig.11.

[0122] The Acinetobacter baumannii bNME7 ortholog was included in this study. A. baumannii, also known as Iraqibacter, is a multidrug resistant bacterium that was brought back to the US by troops returning from Iraq. Infection of cancer patients with A. baumannii was independently associated with increased mortality in a matched case-control study (Nazer et al., 2015). The full-length A. baumanni NME7 was somewhat insoluble, so we also constructed a truncated form with a short, N-terminal deletion (SEQ ID NO: 96). The full-length and truncated A. baumanni NME7s both formed dimers as assessed by size exclusion chromatography. Bacteria NME7 orthologs bind to the extracellular domain of MUC1*

[0123] The recombinantly expressed bNME7s were assayed for binding to the MUC1* ecd by ELISA. For these assays, an ELISA plate was coated overnight at 4°C with a MUC1* ecd peptide coupled BSA at 10 µg / mL in 0.1M carbonate / bicarbonate buffer pH 9.6. After 3 washes with PBS-T (PBS pH 7.4 + 0.05% tween 20), the wells were blocked with 300 µL of PBS-T + 3% BSA for 1h at room temperature. After 3 washes with PBS-T, bacterial NME proteinWSGR Docket No.56699-755.601 (diluted in PBS-T + 1% BSA) was added at different concentration and the plate was incubated 1h at room temperature. After 3 washes with PBS-T, a rabbit polyclonal 6X His tag antibody conjugated to HRP (Abcam, diluted 1:5000 in PBS-T + 1% BSA) was added and the plate was incubated for 45 min at room temperature. Finally, after 3 washes with PBS-T (PBS pH 7.4 + 0.05% tween 20), ABTS solution (Thermofisher) was added and binding was measured at 415 nm.

[0124] Binding of NME7-AB, Hsp593 NME and the truncated A. Baumannii NME7 were compared in an exemplary assay (Fig.12). Unexpectedly, the truncated A. Baumannii NME7 bound to MUC1* ecd with substantially higher affinity than human NME7-AB. Overall, the bNME7s all bound to MUC1* ecd and, in some cases, have stronger binding to MUC1* than human NME7 (Table 12). Table 12. bNME7 binding to the MUC1* ecd as determined by ELISA.WSGR Docket No.56699-755.601

[0125] A consensus sequence of the B3 region of 15 bNME7s that are cancer associated and bind strongly to 43C11 and 39H3 (A415>0.5) is shown in Fig.13A. For comparison, a consensus sequence of the B3 region of 70 bNME7s identified in the BLAST searches is shown in Fig.13B. Antibodies raised against human NME7 cross-react with bacterial NME7 orthologs

[0126] The homology between the human and bacterial NME’s suggested that antibodies against the human NME proteins might cross-react with the bNME7s. To demonstrate cross- reactivity, a panel of anti-human NME antibodies were tested against a panel of bNME7s.

[0127] Three monoclonal antibodies, 4A3, 5D4, and 5A1 raised against the B3 peptide of human NME7 were described in WO2020163325. These antibodies were raised against a variant hNME7 B3 peptide having a serine in place of cysteine and then screened for their ability to bind to human NME7 but not to human NME1. Six additional antibodies, B3C, B3D, B3F, B3A, B3B, and were generated using the same protocol.WSGR Docket No.56699-755.601 Table 13. Antibody sequences (SEQ ID NOs)*Humanized antibodies

[0128] Cross-reactivity of the anti-human NME7 antibodies against bacterial NME7s was determined in an ELISA assay. Polyclonal antibodies against full length NME7 (B-9) and NME1 (C-20) from Santa Cruz Animal Health were included as controls. The recombinant bacterial NMEs and a human NME7-AB control were dispensed into individual wells of a multi- well plate, where they absorbed to the plastic surface. Antibodies that bound to the NMEs were detected using a secondary antibody (anti-mouse or anti-rabbit) coupled to HRP, and absorbance of an HRP substrate (ABTS) was measured at 415 nm. The results shown in Tables 14-15 demonstrate that two of the new anti-human NME7 antibodies, B3C and B3D, cross-react with numerous bNME7s. Table 14. Cross-reactivity of anti-human NME antibodies with bacterial NMEs. Strong binders are highlighted with bold text.WSGR Docket No.56699-755.601* Tuberculosis NME7, but with an R80N mutation as disclosed at pdb 4ANE.WSGR Docket No.56699-755.601 Table 15. bNME7s that bind most strongly to the B3C and B3D anti-human NME7 B3 peptide antibodies.

[0129] A consensus sequence of the B3 region of 15 cancer-associated bNME7s that bound strongly to B3C and B3D (A415>0.5) is shown in Fig.13A. For comparison, a consensus sequence of the B3 region of 70 bNME7s identified in the BLAST searches, without consideration of possible cancer association is shown in Fig.13B.WSGR Docket No.56699-755.601 Epitope mapping of antibodies against the human NME7 B3 peptide

[0130] Epitope mapping was performed to determine which epitopes within the B3 region of human NME7 are most important for antibody cross-reactivity with bNME7s. A series of overlapping peptides spanning the human NME7 B3 region were coupled to BSA through an added C-terminal cystine. To avoid undesired conjugation, a cysteine residue within the native human NME7 sequence was substituted with serine, as shown in (Fig.14). Binding of the peptides to different anti-NME7 monoclonal antibodies was then compared by ELISA, as shown in Fig.15. Epitopes determined from the mapping indicate that the B3C and B3D antibodies that cross-react with bNME7s bind to an epitope with the overlapping sequence QNAVSTD (SEQ ID NO: 93) (Table 16). Within this sequence, the Q is an I, R, E, A, A, H, or M; the V may be an I, the C is a G, and the T is an S in the strongest-binding bNME7s. Thus, B3C binds to a consensus epitope comprising (Q, I, R, E, A, A, H, or M) N A (V or I) H G S D; or more specifically, NAVHGSD (SEQ ID NO: 442). Table 16. Epitopes of anti-human NME7 B3 peptide antibodies.Diagnostic assays for human and bacterial NME7

[0131] Human NME7 can be detected by an immunoassay. Polyclonal or monoclonal antibodies that bind the A domain of NME7 are absorbed to a surface. A sample, which may be derived from blood, cerebrospinal fluid, or tissue, is added, allowing NME7 to bind to the capture antibody. Bound NME7 is detected by a monoclonal antibody that binds to the B domain of NME7. In particular, the antibody may bind to the B3 domain, and may be 4A3, 5A1, or 5D4.

[0132] Fig.16 is a graph comparing the binding of various bNME7s to a polyclonal antibody raised against the B1 peptide of human NME7, as measured by ELISA. These results indicate that the anti-B1 antibody could be used as a capture antibody in a diagnostic assay for bNME7s.WSGR Docket No.56699-755.601 The secondary antibody, which should be more specific, could be an antibody that binds to the B3 peptide region of human NME7 that cross-reacts with bNME7s, such as B3C or B3D, or an antibody raised against a bNME7. NME7 binding to MUC1* in stem cells and cancer cells Cultured human stem cells and cancer cells express full-length NME7 and two forms of NME7 that lack the large N-terminal tandem repeat domain: NME7-AB, a 33kDa polypeptide and NME7-X1, a 30kDa alternatively spliced isoform which is missing an additional 33 N-terminal amino acids when compared to NME7-AB.

[0133] A co-immunoprecipitation experiment was performed to test for NME7 binding to MUC1* in human stem cells. Extracts from induced pluripotent stem cells (iPS7) or embryonic stem cells (HES3) were incubated with an antibody against the MUC1 cytoplasmic tail (Ab-5) or a control antibody (IgG). Immune complexes were co-precipitated and run on a reducing SDS PAGE gel. A western blot from the gel was probed with a commercially available anti-NME7 antibody (B9) (Fig.17A). NME7 bands were detected at ~33kDa and ~30kDa in the immune- precipitates from both stem cell types. The gel was stripped and re-probed with an antibody against the extracellular domain of MUC1* (anti-PSMGFR) to confirm the MUC1* pull-down (Fig.17B). Recombinant NME7-AB and NME7-X1 were mixed, run on a separate gel, and probed with an anti-NME7 antibody (Fig.17C). The NME7 polypeptides that co-precipitated with MUC1* have the same molecular weights as NME7-AB and NME7-X1. These results indicate NME7-AB and NME7-X1 are expressed in stem cells and bind to MUC1*.

[0134] A similar co-immunoprecipitation experiment was performed to test for NME7 binding to MUC1* in T47D breast cancer cells. T47D extracts were incubated with the MUC1 cytoplasmic tail antibody (Ab-5) or a control antibody (IgG). Immune complexes were co- precipitated and run on a reducing SDS PAGE gel. Western blots were probed with two different commercially available anti-NME7 antibodies, B9 (Fig.18A) and 3F7 (Fig.18B). Both blots show NME7 bands at ~33kDa and ~30kDa that co-immunoprecipitated with MUC1* and migrated at the same molecular weights as the NME7 bands from a mixture of NME7-AB and NME7-X1 (Fig.18C). The gels were stripped and re-probed with the anti-MUC1* extracellular domain antibody to confirm that MUC1* was present in the immune-precipitates (Fig.18D-18E). These results indicate NME7-AB and NME7-X1 are expressed in T47D breast cancer cells and bind to MUC1*. NME7 expression in cancer cells and cancer stem cells

[0135] An immunofluorescence experiment was performed to screen for NME7 expression in cells lines representing the major subtypes of cancer that aberrantly express MUC1*. The cellsWSGR Docket No.56699-755.601 were probed with our anti-hNME7 antibody 5A1 and then visualized with an anti-mouse fluorescently labeled secondary antibody (red) and DAPI (blue) (Fig.19A-19T). The results show that hNME7 is expressed by MUC1* positive breast cancer, lung cancer, and pancreatic cancer cells.

[0136] Cancer cell lines and tumors are thought to contain minor subpopulations of cancer stem cells (CSCs) or metastatic cancer cells. We hypothesized that populations of cancer cells can be made more metastatic or increase the proportion of cells that are metastatic by culturing the cells in NME7-AB or NME7-X1. To test that hypothesis, we first cultured MUC1* positive breast cancer T47D cells in standard RPMI media in the presence or absence of anti-NME7 polyclonal antibodies. Three different antibodies were tested: a mixture of A1 and A2 directed toward the NME7 A domain, a mixture of B1 and B2 directed to the NME7 B domain, and antibody #61, which was raised against the NME7 B3 peptide. Antibody #61 can block the binding of NME7 to the extracellular domain of MUC1*. Figs.20A-20B are tables showing that the generation of floating cells, presumably cancer stem cells, is inhibited by the anti-NME7 antibodies. Antibody #61 essentially blocked the generation of the floater cells. Because the table contains scientist observations, two researchers independently viewed the resultant cells and independently estimated the percentage of the cells that were floating. Metastatic cancer cells are characterized by their ability to detach from the primary tumor and maintain viability as separate cells for long periods of time until they implant on tissues that express a chemokine ligand to the chemokine receptor that the metastatic cancer cells express. In the case of breast cancers, metastatic cancer cells upregulate chemokine receptor CXCR4 and tissues to which they can attach and metastasize express CXCL12. In our in vitro assay studying metastatic, or cancer stem cells, “CSCs”, we call these cells the “floaters”. We have also found that the floaters upregulate expression of metastatic markers like CXCR4 as well as stem pluripotency markers, OCT4, NANOG, and SOX2. bNME7 activity in cultured cells

[0137] Recombinant hNME7-AB reverts human stem cells from a Primed state back to a Naïve state. Naïve state stem cells are equivalent to the totipotent cells in the inner cell mass of a blastocyst. Primed state stem cells have acquired cell fate decision marks, such as acetylation and methylation, that limit the types of cells the stem cells can differentiate into. T47D breast cancer cells were cultured in a Minimal Media containing NME7-AB (4nM) and no other growth factor, in Minimal Media containing inhibitors of GSK3-beta and MEK inhibitors “2i”, or in RPMI control media. The 2i inhibitors also revert stem cells back to a naïve state (Nichols & Smith, 2011).WSGR Docket No.56699-755.601

[0138] In some of the wells, anti-NME7 antibody #61 was added to block the interaction of NME7 with MUC1*. Antibody #61 binds to the B3 peptide of hNME7. Fig.21 shows the amount of RNA, indicative of growth, that is present after 10 days in culture under each condition. The table shows that in the presence of anti-hNME7 antibody #61, there is little to no growth of the T47D cells. Fig.22 shows a graph of the expression of CXCR4, measured in a PCR assay, for each condition. Note that even though we have not added exogenous NME7 to the 2i condition, the addition of the anti-hNME7 B3 antibody inhibited the upregulation of CXCR4. The arrows point from T47D cancer cell grown in 2i minimal media to 2i minimal media plus anti-hNME7 antibody; the fold increase in CXCR4 expression, compared to growth in RPMI media falls from a 10-fold increase to zero in the presence of the antibody. The second arrow points from the cancer cells grown in hNME7-AB, where CXCR4 is expressed 70-fold higher than the control grown in RPMI media, to where CXCR4 fold increase in expression drops to only 10-fold.

[0139] Note that even though we have not added exogenous NME7 to the 2i condition, the addition of the anti-hNME7 B3 antibody inhibited the upregulation of CXCR4. To investigate, we measured the expression of metastatic markers as well as stem cell pluripotency markers for T47D breast cancer cells grown in Minimal Media alone compared to standard serum containing RPMI media (Fig.23A-23D). Fig.23A is a graph of a PCR experiment in which T47D breast cancer cells that were cultured for 10 days in minimal media alone have increased expression of hNME7 and the alternative splice isoform hNME7-X1. Fig.23B is a graph of a PCR experiment in which T47D cells cultured in minimal media alone have increased expression of MUC1. As can be seen in the figure, serum starving cancer cells, that is to say growth in Minimal Media alone, induces upregulation of hNME7, NME7-X1 and MUC1. The induced expression of the hNME7s and MUC1 likely accounts for some of the hNME7-like biological function observed in minimal media conditions. However, growth in human or bacterial NME7s, in general, out-performed minimal media alone.

[0140] Human T47D breast cancer cells were also grown minimal media supplemented with various bNME7s having high sequence homology to the B3 peptide region of hNME7. PCR was performed to see if, like hNME7, growth in bNME7s induced upregulation of metastatic markers, such as CXCR4 for breast cancers or stem cell pluripotency markers, like SOX2, OCT4 and NANOG Fig.24A-24D. As can be seen in the figure, metastatic marker CXCR4 and pluripotency markers SOX2, OCT4 and NANOG have increased expression, especially in the floater population, when cultured in bNME7s compared to the same cell grown in RPMI.

[0141] We selected 11 bNME7s for more in-depth biological function assays, as a demonstration. These bNME7s included: IB2 (A. baumannii); H3-1 (HalomonasWSGR Docket No.56699-755.601 heilongjiangensis); A2-4 (Gammaproteobacteria bacterium); HSP-593 (Halomonas so.593); 21MCB17 (Gammaproteobacteria bacterium); 21MBM31 (Chlamydiae bacterium); D4-1 (Perlabentimonas gracilis); C1-2 (Neisseria); F1-1 (Betaproteobacteria bacterium); and E3-3 (Neisseria baciliformis); E2-3 (Deltaproteobacteria bacterium). hNME7-AB and MM, minimal media were included as controls, although recall that growth of cancer cells in minimal media alone induced expression of hNME7, hNME7-X1 and MUC1. The 11 were chosen because of their diversity. Some bound to MUC1* with a higher affinity than hNME7-AB, while others bound poorly to the peptide. Some were dimers while others were tetramers or hexamers, recalling that MUC1* extra cellular domain must be dimerized to be activated. The percent sequence identity to hNME7 of the group of 11 also varied.

[0142] In view of the fact that hNME7-AB can support the growth of human stem cell in the naïve state, we tested bNME7s for their ability to grow and maintain pluripotency while in the naïve state. Each recombinant bNME7 was added to a Minimal Media, “MM” to a final concentration of 4nM, assuming each was a dimer. In other experiments hNME7 and bNME7s were added at concentrations ranging from 1 nM to 36 nM. In this specific experiment, they were added at 4 nM where the molecular weight used was that of the dimer. Human induced pluripotent stem cells were first cultured in huNME7-AB, which maintains stem cells in the naïve state. The stem cells were then plated onto gelatin to avoid any biological signaling through surface adhesion coatings such as MEFs, mouse embryonic fibroblasts, Matrigel, and the like. Additionally, it is known that only naïve state stem cells can grow on gelatin (Ortmann et al., 2020). Immediately after plating, stem cells were rinsed and to each well a bNME7 in minimal media was added. Minimal Media was included as a control. Media was changed every 24 hours. Stem cells were imaged that documented stem cell morphology. Stem cells were periodically assayed for expression of pluripotency marker OCT4 as well as for naïve state marker antibody against H3K27me, that stains stem cells in which both X chromosomes are still active. Human stem cells cultured in various bNME7s robustly grew and resisted spontaneous differentiation for up to 144 hours. The bacterial NME7s induced robust stem cell growth and pluripotency as well as the naïve state over a 144-hour period. Fig.25A is a graph measuring the percent confluency of the stem cells, as an indicator of growth induced by the bNME7s. Percent confluence was measured digitally using Image J on photographs of the stem cells 96 hours post addition of the bNME7s. Fig.25B is a graph of OCT4 positivity, indicating the pluripotency of the stem cell population. OCT4 was measured by digital image analysis of integrated pixel density of stem cells stained with an OCT4 antibody bearing a green fluorescent tag. Fig.25C is a graph of H3K27me, indicating the X chromosome activation status. H3K27me was measured by digital image analysis of integrated pixel density of stem cells stained with an H3K27meWSGR Docket No.56699-755.601 antibody bearing a red fluorescent tag. The first step of human stem cell differentiation is, in female source cells, inactivation of one of the X chromosomes. A fluorescently labeled antibody that detects a tri-methylated Lysine 27 on Histone 3 H3K27me looks like a red cloud-like pattern over the entire nucleus when both X chromosome are still active. Once one of the X chromosomes is inactivated, the diffuse cloud-like stains changes and appears as a condensed, punctate red stain in a small part of the nucleus.

[0143] Fig.26 shows magnified images of representative stem cells in this experiment along with the digital scoring system used in graphs comparing the bNME7s to hNME7 and to Minimal Media as a control. Figs.26A-26H show photographs of stem cells stained for the expression of OCT4 and a scoring system from 1 to 4 for assessing pluripotency, where 1 is the highest and 4 is the lowest. The portions indicated by dotted line indicates areas of cells where OCT4 intensity is reduced by 50%, 75% or 100% compared to the typical bright OCT4 staining. Figs.26I-26P show photographs of stem cells stained for the expression of H3K27me that indicates that both X chromosomes are active or where one X has been inactivated. The photographs and corresponding scoring system from 1 to 4 for assessing XX activation, where 1 is the highest and 4 is the lowest. Fig.26Q shows a graph of the percentages of stem cells that are 100% pluripotent and OCT4 positive, or at various stages of differentiation as detailed in the scoring scheme above the graph. Fig.26R shows a graph of the percentages of stem cells that are 100% XaXa, meaning both X chromosomes are still active, which is called the naïve state, or at various stages of differentiation toward inactivating one X, as detailed in the scoring scheme above the graph. Fig.27 is a table summarizing the ability of the bNME7s to promote stem cell growth and their corresponding binding to MUC1* relative to that of hNME7-AB. The table also shows their multimerization state as determined by size exclusion chromatography. Here we note that the bNME7s that resulted in the highest rates of stem cell growth were dimers. It is possible that at lower concentrations than those measured by SEC, the other bNME7s could also exist as dimers, although the bNME7s were added to minimal media for stem cell growth to a final concentration of 4 nM, with molecular weight used was that of the dimer. Fig.28 shows the table of dimers only, reflecting a near perfect correlation between strength of binding to MUC1* extra cellular domain peptide and growth rate of stem cells cultured in that bNME7. bNME7s can activate MUC1* to generate cancer stem cells and support their metastasis

[0144] Cancer cells are more metastatic in xenograft experiments after growth in hNME7-AB. T47D breast cancer cells were grown for 10 days in a serum-free minimal media containing recombinant hNME7-AB as the only growth factor. About 25% of the cancer cells began floating when grown in hNME7-AB. PCR measurements showed that these “floating” cellsWSGR Docket No.56699-755.601 greatly upregulated expression of stem cell pluripotency markers as well as the breast cancer metastatic factor CXCR4. These floater cells are thought to be cancer stem cells (CSCs). The test of a cancer stem cell, also known as a tumor initiating cell, is if they can form a tumor in a test mouse from a small number of cells, which is less than the millions of cells used in typical xenograft experiments. Immune compromised female nu / nu mice were implanted with 90-day release estrogen pellets. Either 500,000 T47D parent cells or 10,000 of the hNME7-AB grown T47D floater CSCs were injected into the tail vein of the nu / nu mice. These cancer cells were engineered to express Luciferase. To visualize the cancer cells, animals are injected with Luciferin, then visualized on an IVIS instrument 10 minutes later. As can be seen in the IVIS measurements of Figs.29A-29E, by Day 10 the 500,000 parent T47D cells injected into the tail vein show little to no signs of cancer or metastasis (Fig.29A). In stark contrast, the 10,000 of the hNME7-AB grown T47D-CSCs injected into the tail vein have metastasized in both of the two test mice (Fig.29B-29C). One of the two metastatic mice (Fig.29C) was then treated with a cocktail of anti-NME7 monoclonal antibodies 5A1, 4A3, and 5D4 that bind to the NME7 B3 peptide, at a concentration that corresponds to 15 mgs / kg. By Day 12, the untreated mice had died of total cancer metastasis. In contrast, the treated mouse has begun to clear the cancer (Fig. 29D). By Day 14 the mouse just has a few residual cancer cells (Fig.29E). At that time anti- NME7 antibody treatment was paused to see if the cluster of cancer cells could continue to metastasize. By Day 21, that mouse is almost totally metastatic again. Anti-NME7 antibody treatment was resumed with two additional doses. The animal was again cleared of cancer and showed no signs of ill health until Day 31 when the experiment was ended (Fig.29F-29N). The animal injected with 50-times more of the T47D parent cells never metastasized (Fig.29O- 29W).

[0145] A similar experiment was carried out wherein T47D breast cancer cells were cultured for 10 days in a bNME7. PCR measurements of metastatic marker CXCR4 as well as stem cell pluripotency genes are shown in Figs.24A-24D. Immune compromised female nu / nu mice were implanted with 90-day release estrogen pellets. Either 300,000 T47D parent cells or 30,000 of the bNME7 grown T47D floater CSCs were injected into the tail vein of the nu / nu mice. Twice per week, each mouse was i.v. injected with the recombinant bNME in which the injected CSCs had been grown. As can be seen in the IVIS measurements of Figs.30A-30G, by Day 18 the mice injected with bNME7 grown cells have induced metastasis from injection of only 30,000 cells. By Day 18, metastases had formed in mice injected with T47D CSCs activated by recombinant NME7 proteins from A. baumannii (Fig.30A), Halomonas heilongjiangensis (Fig. 30B), Gammaproteobacteria bacterium (Fig.30C), human (Fig.30D), Halomonas sp.593 (Fig. 30E), Gammaproteobacteria bacterium (Fig.30F) and Chlamydiae bacterium (Fig.30G). TheseWSGR Docket No.56699-755.601 results demonstrate that bNME7 grown cancer cells induced metastasis from injection of only 30,000 cells.

[0146] Methods: 10,000 - 30,000 T47D Floater cells, “CSCs”, were injected into the tail vein of female nu / nu mice.300,000 of the parent T47D cells, grown in RPMI media were injected into the tail vein of other nu / nu mice. The cancer cells had been engineered to express Luciferase and cancer cells within the mice were tracked by bioluminescence measurement on an IVIS instrument. In some cases, the human or bacterial NME7s were also injected after the cancer cell implantation. Design of bNME7 peptides and consensus sequences for vaccines and antibody production

[0147] Based in part on the results of our testing of bNME7 mimicry of hNME7 biological function, consensus sequence peptides were designed for use in producing anti-cancer antibodies as well as for their incorporation into vaccines, either individually or as combinations (Fig.31). In some cases, it would be desirable to make a vaccine that would be effective against bNME7s as well as hNME7. In other cases, it would be desirable to make a vaccine that would be more effective against bNME7s than hNME7.

[0148] As can be seen in Fig.31, the first set of consensus sequence peptides, CANBAC1, CANBAC2 and CANBAC3 (SEQ ID NOs: 82-84) are likely to be effective against both hNME7 and bNME7s, as indicated by sequence identity of 45% - 75% and E- values of E-07 to E-17, with the highest sequence identity and E- values for hNME7.

[0149] The next set of consensus sequence peptides CANBAC4 – CANBAC9 (SEQ ID NOs: 416-421) replaces the human NME7 sequence of NAVHCTD (SEQ ID NO: 444) with consensus sequences more conserved in bacteria: NAVHGSD (SEQ ID NO: 442) or NAAHGSD (SEQ ID NO: 443). This group is likely to be more effective against bNME7s, as sequence identity for hNME7 dropped to as low as 37% and E- values worsen to E-7 to E-10. Conversely, sequence identity for bNME7s for this CANBAC group increase to over 90% and E- values improve to between E-12 and E-18.

[0150] In the next set of consensus sequence peptides CANBAC10 – CANBAC18 (SEQ ID NOs: 422-430), we sought to increase specificity to both bacterial and human NMEs that share sequence identity in the region of the B3 peptide but where the N-terminus has been extended to include the conserved “GTIR” (SEQ ID NO: 448) common to bNME7s that function like hNME7. Peptides that include GTIR as well as NAVHGSD (SEQ ID NO: 442), NAAHGSD (SEQ ID NO: 443) or NAVHCTD (SEQ ID NO: 449) share up to 75% sequence identity to the human sequence with E- values of E-19. The bNME7s share up to 93% sequence identity and have improved E- values, such as 6.00 E-23.WSGR Docket No.56699-755.601

[0151] Consensus sequence peptides CANBAC19 – CANBAC22 (SEQ ID NOs: 431-434) are shorter peptides focused on the region where bNME7s have the least sequence identity to hNME7. These peptides are expected to induce expression of antibodies that bind to bNME7s more than hNME7 for use in generating antibodies or for incorporation into vaccines.

[0152] Consensus sequence peptides CANBAC23 – CANBAC29 (SEQ ID NO: 435-441) are designed to generate antibodies that bind to bNME7s more than hNME7 because they are extended at the C-terminus where there is little to no sequence identity to the human sequence KILDN (SEQ ID NO: 550). Percent sequence identity is lower for hNME7 and higher for bNMEs, with corresponding E- values. Methods

[0153] Minimal media, abbreviated here as “MM” is generally a serum free media that contains a serum replacement plus some non-essential amino acids. Although the concentrations can vary, the formulation used here is: 400 ml DMEM / F12 / GlutaMAX I (Invitrogen# 10565- 018), 100 ml Knockout Serum Replacement (KO-SR, Invitrogen# 10828-028), 5 ml 100x MEM Non-essential Amino Acid Solution (Invitrogen# 11140-050) and 0.9 ml (0.1mM) β- mercaptoethanol (55mM stock, Invitrogen# 21985-023). The DMEM media can be substituted for any other base media.

[0154] For testing recombinant bacterial NME7s for ability to maintain pluripotency and inhibit differentiation, bacterial NME7s, were generated and purified. Human female induced pluripotent stem cells (iPSC) were grown in 4nM NME7-AB containing medium in 6-well cell culture plates (VitaTM, Thermo Fisher) that had been coated with 12.5 ug / well of a monoclonal anti-MUC1* antibody MN-C3 for 3-4 passages. Cells were plated at a density of 300,000 cells per well.

[0155] The iPS cells were passaged using Accutase (Life Technologies #A11105-01) and resuspended in base media containing either 4nM of NME7-AB or bacterial NME7s. iPS cells were plated at 12,500 - 25,000 cells per well in 12-well plates (Nunclon DeltaTM, ThermoFisher) coated with 0.2% gelatin. Media was changed every 48 hours and cells were cultured for 192 hours. Comparable pluripotent stem cell growth was achieved when stem cells were grown in bacterial NME or in NME7-AB containing media.

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PLOS ONE 8(3): e57867. https: / / doi.org / 10.1371 / journal.pone.0057867 57. Nazer LH, Kharabsheh A, Rimawi D, Mubarak S, Hawari F. Characteristics and Outcomes of Acinetobacter baumannii Infections in Critically Ill Patients with Cancer: A Matched Case-Control Study. Microb Drug Resist.2015 Oct;21(5):556-61. doi: 10.1089 / mdr.2015.0032. 58. Nichols J, Smith A. The origin and identity of embryonic stem cells. Development.2011 Jan;138(1):3-8. doi: 10.1242 / dev.050831. PMID: 21138972.WSGR Docket No.56699-755.601 59. Ortmann D, Brown S, Czechanski A, Aydin S, Muraro D, Huang Y, Tomaz RA, Osnato A, Canu G, Wesley BT, Skelly DA, Stegle O, Choi T, Churchill GA, Baker CL, Rugg-Gunn PJ, Munger SC, Reinholdt LG, Vallier L. Naive Pluripotent Stem Cells Exhibit Phenotypic Variability that Is Driven by Genetic Variation. Cell Stem Cell.2020 Sep 3;27(3):470- 481.e6. doi: 10.1016 / j.stem.2020.07.019.

Claims

WSGR Docket No.56699-755.601 CLAIMS We claim:

1. An NME7 vaccine comprising a peptide comprising: a. a sequence of SEQ ID NO: 442 or a fragment and / or variant thereof, b. a sequence of SEQ ID NO: 443 or a fragment and / or variant thereof, c. a sequence of SEQ ID NO: 444 or a fragment and / or variant thereof, d. a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, e. a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, f. a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, g. a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof h. a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, i. a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, j. a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, k. a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, l. a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, m. a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, n. a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, o. a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, p. a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof,WSGR Docket No.56699-755.601 q. a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, r. a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, s. a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, t. a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, u. a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, v. a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof, or w. the sequence of SEQ ID NO:

448.

2. The NME7 vaccine of claim 1, wherein the peptide comprises at least 8, 10, or 12 amino acids.

3. The NME7 vaccine of claim 1 or claim 2, wherein the peptide comprises no more than 50 amino acids.

4. The NME7 vaccine of claim 1 or claim 2, wherein the peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids.

5. The NME7 vaccine of any one of claims 1-4, wherein the variant comprises a substitution mutation.

6. The NME7 vaccine of any one of claims 1-4, wherein the variant comprises two or three substitution mutations.

7. The NME7 vaccine of any one of claims 1-6, wherein the peptide a. has higher sequence identity to a peptide of human NME7 than to any peptide of human NME1, or b. the peptide has less than 40% sequence identity to any peptide of human NME1.

8. The NME7 vaccine of any one of claims 1-7, further comprising a second NME7 peptide.

9. The NME7 vaccine of claim 8, wherein the second NME7 peptide comprisesWSGR Docket No.56699-755.601 a. SEQ ID NO: 81 or a fragment and / or variant thereof, b. a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof, c. a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, d. a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, e. a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, f. a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, g. a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, h. a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, i. a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, j. a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, k. a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, l. a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, m. a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, n. a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, o. a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, p. a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, q. a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof,WSGR Docket No.56699-755.601 r. a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, s. a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, t. a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, or u. a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof.

10. The NME7 vaccine of claim 9, wherein: a. the second NME7 peptide comprises at least 8, 10, or 12 amino acids, b. the second NME7 peptide comprises no more than 50 amino acids, c. the second NME7 peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids, d. the variant comprises a substitution mutation, and / or e. the variant comprises two or three substitution mutations.

11. The NME7 vaccine of any one of claims 1-10, wherein the vaccine further comprises a carrier protein, adjuvant, or immunogenic agent.

12. The NME7 vaccine of any one of claims 1-11, wherein the peptide is coupled to the carrier protein, adjuvant or immunogenic agent.

13. The NME7 vaccine of any one of claims 1-12, wherein the peptide is connected to another peptide sequence via a spacer or linker.

14. The NME7 vaccine of any one of claims 1-13, further comprising a pharmaceutically acceptable carrier.

15. The NME7 vaccine of any one of claims 1-14, wherein an NME7 vaccine is formulated in a dosage unit form.

16. The NME7 vaccine of any one of claims 1-15, wherein the vaccine is an anti-cancer vaccine.

17. A nucleic acid encoding the peptide or fragment and / or variant thereof of claim 1.

18. The nucleic acid of claim 17, wherein the nucleic acid is operatively linked to an expression control sequence.WSGR Docket No.56699-755.601 19. A method of isolating an anti-NME7 antibody comprising: selecting an antibody that binds to: a. an epitope comprising SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or b. an antigenic peptide comprising: i. a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof, ii. a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, iii. a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, iv. a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, v. a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof vi. a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, vii. a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, viii. a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, ix. a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, x. a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, xi. a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, xii. a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, xiii. a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, xiv. a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof,WSGR Docket No.56699-755.601 xv. a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, xvi. a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, xvii. a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof, xviii. a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, xix. a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, or xx. a sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof; and determining that the antibody binds to an NME7.

20. The method of claim 19, wherein: a. the antigenic peptide comprises at least 8, 10, or 12 amino acids, b. the antigenic peptide comprises no more than 50 amino acids, c. the antigenic peptide comprises no more than 40, 35, 33, 29, 24, 20, 19 or 15 amino acids, d. the variant comprises a substitution mutation, and / or e. the variant comprises two or three substitution mutations.

21. The method of claim 19 or claim 20, further comprising injecting an animal with the peptide or an NME7 polypeptide comprising the peptide.

22. The method of any of claims 19-21, further comprising: a. determining that the anti-NME7 antibody binds to an NME7 with higher affinity than it binds to NME1, b. determining that the anti-NME7 antibody binds to a bacterial NME7 with higher affinity than it binds to human NME7, or c. determining that the anti-NME7 antibody inhibits the binding of an NME7 to MUC1*.

23. An antibody that binds to an NME7 polypeptide comprising: a. a sequence selected from SEQ ID NO: 442, SEQ ID NO: 443, or SEQ ID NO: 444, or a fragment and / or variant thereof,WSGR Docket No.56699-755.601 b. a sequence selected from SEQ ID NOs: 180, 195, 249, 239, 187, 204, 213, 244, 256, 230, 242, 185, 181, 253, 182, 233, 250, 251, 243, 252, 227, 191, 220, 212, 208, 196, 219, 200, 186, 209, 255, 226, and 188 or a fragment and / or variant thereof, c. a sequence selected from SEQ ID NOs: 179-257 or a fragment and / or variant thereof, d. a sequence selected from SEQ ID NOs: 256-336 or a fragment and / or variant thereof, e. a sequence selected from SEQ ID NOs: 336-415 or a fragment and / or variant thereof, f. a sequence selected from SEQ ID NOs: 82-84 or a fragment and / or variant thereof, g. a sequence selected from SEQ ID NOs: 416-421 or a fragment and / or variant thereof, h. a sequence selected from SEQ ID NOs: 422-430 or a fragment and / or variant thereof, i. a sequence selected from SEQ ID NOs: 431-434 or a fragment and / or variant thereof, j. a sequence selected from SEQ ID NOs: 435-441 or a fragment and / or variant thereof, k. a sequence selected from SEQ ID NOs: 179, 258, and 337 or a fragment and / or variant thereof, l. a sequence selected from SEQ ID NOs: 180, 259 and 339 or a fragment and / or variant thereof, m. a sequence selected from SEQ ID NOs: 249, 328 and 407 or a fragment and / or variant thereof, n. a sequence selected from SEQ ID NOs: 185, 264 and 343 or a fragment and / or variant thereof, o. a sequence selected from SEQ ID NOs: 204, 283 and 362 or a fragment and / or variant thereof, p. a sequence selected from SEQ ID NOs: 222, 301 and 380 or a fragment and / or variant thereof, q. a sequence selected from SEQ ID NOs: 233, 312 and 391 or a fragment and / or variant thereof,WSGR Docket No.56699-755.601 r. a sequence selected from SEQ ID NOs: 201, 280 and 359 or a fragment and / or variant thereof, s. a sequence selected from SEQ ID NOs: 244, 323 and 402 or a fragment and / or variant thereof, or t. sequence selected from SEQ ID NOs: 252, 331 and 410 or a fragment and / or variant thereof.

24. The antibody of claim 23, wherein a. the antibody binds to a human or bacterial NME7 with higher affinity than it binds to human NME1, and / or b. the antibody binds to a bacterial NME7 with higher affinity than it binds to human NME7.

25. An anti-NME7 antibody comprising three heavy chain (HC) complementarity determining regions (CDR) and three light chain (LC) CDRs, wherein: a. HC-CDR1 comprises SEQ ID NO: 41, HC-CDR2 comprises SEQ ID NO: 42, HC-CDR3 comprises SEQ ID NO: 43, LC-CDR1 comprises SEQ ID NO: 44, LC-CDR2 comprises SEQ ID NO: 45, and LC-CDR3 comprises SEQ ID NO: 46; b. HC-CDR1 comprises SEQ ID NO: 47, HC-CDR2 comprises SEQ ID NO: 48, HC-CDR3 comprises SEQ ID NO: 49, LC-CDR1 comprises SEQ ID NO: 50, LC-CDR2 comprises SEQ ID NO: 51, and LC-CDR3 comprises SEQ ID NO: 52; c. HC-CDR1 comprises SEQ ID NO: 53, HC-CDR2 comprises SEQ ID NO: 54, HC-CDR3 comprises SEQ ID NO: 55, LC-CDR1 comprises SEQ ID NO: 56, LC-CDR2 comprises SEQ ID NO: 57, and LC-CDR3 comprises SEQ ID NO: 58; d. HC-CDR1 comprises SEQ ID NO: 59, HC-CDR2 comprises SEQ ID NO: 60, HC-CDR3 comprises SEQ ID NO: 61,WSGR Docket No.56699-755.601 LC-CDR1 comprises SEQ ID NO: 62, LC-CDR2 comprises SEQ ID NO: 63, and LC-CDR3 comprises SEQ ID NO: 64; or e. HC-CDR1 comprises SEQ ID NO: 65, HC-CDR2 comprises SEQ ID NO: 66, HC-CDR3 comprises SEQ ID NO: 67, LC-CDR1 comprises SEQ ID NO: 68, LC-CDR2 comprises SEQ ID NO: 69, and LC-CDR3 comprises SEQ ID NO:

70.

26. The antibody of any one of claims 23-25, wherein the antibody is humanized.

27. The antibody of any one of claims 23-26, wherein the antibody inhibits the binding of an NME7 to MUC1*.

28. A pharmaceutical composition comprising the antibody of any one of claims 23-27.

29. A nucleic acid encoding the antibody of any one of claims 23-27.

30. An expression vector comprising a promoter and the nucleic acid of claim 29.

31. A host cell comprising the expression vector of claim 30.

32. A diagnostic assay for detecting a bacterial NME7 ortholog (bNME7) comprising contacting a sample with the antibody of any one of claims 23-27.

33. The diagnostic assay of claim 32, further comprising contacting the sample with a second anti-NME7 antibody.

34. The diagnostic assay of claim 33, wherein the second anti-NME7 antibody binds to an epitope in the B1 region of the bNME7.

35. The diagnostic assay of claim 34, wherein the second antibody is a polyclonal antibody.

36. The diagnostic assay of any one of claims 29-35, wherein sample is a blood sample, a plasma sample, or a serum sample.

37. The diagnostic assay of any one of claims 29-35, wherein the sample is a tumor biopsy sample.WSGR Docket No.56699-755.601 38. A method of treating a MUC1* positive cancer in a subject comprising administering to the subject the pharmaceutical composition of claim 28.

39. A method of preventing or treating a MUC1* positive cancer in a subject comprising administering to the subject the vaccine of any one of claims 1-16 or the nucleic acid of claim 17 or claim 18.

40. The method of claim 39, wherein the method inhibits metastasis of the cancer.

41. A method of preventing a MUC1* positive cancer in a subject comprising: a. determining that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7, and b. administering to the subject the vaccine of any one of claims 1-16 or the nucleic acid of claim 17 or claim 18.

42. A method of reducing the risk of a MUC1* positive cancer in a subject comprising: a. determining that the subject comprises or is infected with a bacteria expressing a polypeptide with at least 50% sequence identity to human NME7, and b. administering an antimicrobial agent to the subject; thereby killing the bacteria.

43. A method of determining that a subject is at increased risk of a MUC1* positive cancer comprising identifying a microorganism that expresses an NME7 polypeptide in a sample from the subject, wherein the NME7 polypeptide comprises a sequence with at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to human NME7 or the B3 peptide of human NME7.

44. The method of claim 43, further comprising: a. determining if the bacterial NME7 binds to the extracellular domain of MUC1*, b. determining if the bacterial NME7 stimulates stem cell growth or maintains stem cell pluripotency, and / or c. determining that the bacterial NME7 forms a dimer.

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