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WO2024249713A2 - Inhibition de métastase avec des produits végétaux recombinants - Google Patents

Inhibition de métastase avec des produits végétaux recombinants Download PDF

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WO2024249713A2
WO2024249713A2 PCT/US2024/031797 US2024031797W WO2024249713A2 WO 2024249713 A2 WO2024249713 A2 WO 2024249713A2 US 2024031797 W US2024031797 W US 2024031797W WO 2024249713 A2 WO2024249713 A2 WO 2024249713A2
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seq
remsl1
emsl1
protein
glycans
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WO2024249713A3 (fr
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Amy M. FULTON
David J. Weber
Raquel GODOY-RUIZ
Namita Kundu
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University of Maryland Baltimore
US Department of Veterans Affairs
University of Maryland College Park
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University of Maryland Baltimore
US Department of Veterans Affairs
University of Maryland College Park
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention described herein relates to the field of medicine, and in particular, to cancer and cancer metastasis treatment.
  • a new compound has been identified as a recombinant protein with potent anti-metastatic activity, similar to the anti-metastatic activity for breast cancer previously reported for taro plant from Colocasia esculenta.
  • This new compound is identified as recombinant Engineered Mannose-Specific Lectin 1 (rEMSL1) family.
  • rEMSL1 Engineered Mannose-Specific Lectin 1
  • TNBC triple negative breast cancer
  • taro extract A water-soluble, heat labile, high molecular weight (>5 kDa) extract of raw taro, termed taro extract (TE), has the ability to inhibit metastasis in two pre-clinical models of TNBC. This protective activity is destroyed in cooked Taro.
  • taro extract from Colocasia esculenta classified proteins into two major groups: two albumins, A1 and A2 (molecular masses (molecular mass 12-14 kDa and 55-60 kDa, respectively), and two globulins, G1 and G2 (molecular masses 14 k Da and 22- 24 kDa, respectively).
  • G1 was identified by N-terminal sequencing and clustered as G1a/G1c and G1b/G1d, C- and N-terminal subdomains, respectively, derived from pro-proteins. Microheterogeneity of lectins within the GNA-related superfamily has been reported. In particular, it has been reported that each subgroup was formed by several polypeptides, and that ten isoforms (pI range 5.5 to 9.5) exist.
  • GNA-related lectins superfamily mostly exists as complex mixtures of 12-14 kDa polypeptides, which have been identified by 2D electrophoresis with apparent molecular masses of 12 kDa (pI range 6.5-9). Ten of these polypeptides have been identified as lectin (A5HMM7) isomers by MALDI-TOF mass spectrometry. Phylogenetic analysis of the available sequences indicated that these proteins most probably result from multiple independent domain duplication/in tandem insertion events.
  • G2 group globulins are composed of two groups of proteins, G2a (molecular mass 24 kDa) and G2b (molecular mass 22 kDa), with a native molecular mass of 50 kDa.
  • G2a molecular mass 24 kDa
  • G2b molecular mass 22 kDa
  • Phylogenetic analysis of G2 sequences has shown that they belong to the Kunitz family of trypsin/chymotrypsin inhibitors, which includes the sweet potato storage protein sporamin. These activities may also indicate that these proteins may have evolved from ancestral proteins with metabolic activity.
  • the polypeptide composition of taro extracts can change slightly among distinct cultivars, as demonstrated by electrophoresis analyses.
  • the present invention relates to therapeutic agent(s) derived from edible roots of the plant Colocasia esculenta, commonly known as Taro.
  • the invention in some embodiments, relates to a recombinant taro storage protein derivative.
  • FIG.1A is an SDS-PAGE of untagged proteins (pET24 expression vector): rEMSL2 D11 (26.9 kDa) (SEQ ID NO:38; #211), rEMSL8 D11 (27.7 kDa) (SEQ ID NO:54; #811), and rEMSL1 D11 (29.4 kDa) (SEQ ID NO:23; #011) overexpressed in BL21(DE3) and Rosetta cells.
  • FIG.1B is an SDS-PAGE of Sumo-fusion engineered proteins (pSumo expression vector): rEMSL2 D31 (37 kDa) (SEQ ID NO:46; #231), rEMSL8 D31 (38 kDa) (SEQ ID NO:62; #831), and rEMSL1 D31 (39 kDa) (SEQ ID NO:31; #031) overexpressed in BL21(DE3) and Rosetta cells.
  • FIG.1C is an SDS-PAGE of untagged proteins (pET24 expression vector): rEMSL2 D11 (26.9 kDa) (SEQ ID NO:38; #211), rEMSL8 D11 (27.7 kDa) (SEQ ID NO:54; #811), and rEMSL1 D11 (29.4 kDa) (SEQ ID NO:23; #011) overexpressed in BL21(DE3) and Rosetta cells.
  • FIG.1D is an SDS-PAGE of Sumo-fusion engineered proteins (pSumo expression vector): rEMSL2 D31 (37 kDa) (SEQ ID NO:46; #231), rEMSL8 D31 (38 kDa) (SEQ ID NO:62; #831), and rEMSL1 D31 (39 kDa) (SEQ ID NO:31; #031) overexpressed in BL21(DE3) and Rosetta cells.
  • FIG.1E shows the purification of 15 N-labeled recombinant Mannose Specific Lectin 1 (rEMSL1 D1 (1-268)) (SEQ ID NO:9; #001) size exclusion (S200-PG) column chromatogram expressed using pSUMO expression vector (SEQ ID NO:31; #031).
  • FIG.1F is an SDS-PAGE of fractions 8-16 eluted from the S200 column.
  • FIG.1G provides the amino acid sequence corresponding to rEMSL1 D1 (SEQ ID NO:9; #001).
  • FIG.1H shows the 1 H- 15 N TROSY of 15 N-labeled rEMSL1 D4 (residues, 28-253) by Nuclear Magnetic Resonance (NMR results) (SEQ ID NO:22; #004).
  • FIG.1I shows that rEMSL1 D11 (residues 1-268) theoretical monoisotopic mass is 29542.75 Da if all cysteines are reduced (SEQ ID NO:9; #001) [0023]
  • FIG.1J provides the sequence of EMSL1 D1 (SEQ ID NO:23;#011)
  • FIG.1K is an SDS-PAGE of 1.(eGFP)-EMSL2 D23 (residues 140-216) (41,8 kDa) (SEQ ID NO.44;#223); 2. (eGFP)-EMSL2 D22 (residues 24-122) (39,3 kDa) (SEQ ID NO. 43;#222); 3.
  • eGFP EMSL1 D23 (residues 145-240) (41,7 kDa) (SEQ ID NO.29;#023); 4.
  • eGFP EMSL1 D22 (residues 29-127) (42,3 kDa) (SEQ ID NO.28;#022); 5.
  • eGFP EMSL1 D23 (residues 145-240) (41,7 kDa) (SEQ ID NO.29;#023); 4.
  • eGFP EMSL1 D22 (residues 29-127) (42,3 kDa) (SEQ ID NO.28;#022); 5.
  • eGFP EMSL1 D23 (residues 145-240) (41,7 kDa) (SEQ ID NO.29;#023); 4.
  • eGFP EMSL1 D22 (residues 29-127) (42,3 kDa
  • FIG.2A shows TE size exclusion chromatography Superdex S200 chromatographic data. Column molecular weight calibration was performed with protein standards as described in “Methods” section.
  • FIG.2B shows TE proteins resolved in SDS-PAGE (4-20%) on mainly 3 bands corresponding to 12, 23 and 52 kDa.
  • FIG.2C shows an intact mass spectrum of a TE sample by MS-ESI.
  • FIG.3A, FIG.3B, and FIG.3C shows MALDI-TOF-MS results for TE glycopeptides of 12 kD , 23 kD and 52 kD MALDI-TOF-MS, respectively.
  • FIG.4A shows the multiple sequence alignment of EMSL1 (Q39487), EMSL2 (A5HMM7), EMSL3 (PDBID 5T20), EMSL5 (R9RL27), EMSL8 (Q43418) obtained using MUSCLE.
  • FIG.4B shows the pairwise sequence alignment between ETCI1 (Q39488), ETCI2 (Q39489).
  • FIG.4C shows a Neighbor Joining tree calculated from the multiple sequence alignment (FIG.4A-4A and FIG.4B-4B) of EMSL1 (SEQ ID NO:9; #001), EMSL2 (SEQ ID NO:10; #201), EMSL3 (SEQ ID NO:11; #301), EMSL5 D1 (SEQ ID NO:13; #501), EMSL5 D10 (SEQ ID NO:50; #510), EMSL8 (SEQ ID NO:16; #801), ECTI1 (SEQ ID NO:71; #1101), and ECTI2 (SEQ ID NO:72; #1201) proteins.
  • EMSL1 SEQ ID NO:9; #001
  • EMSL2 SEQ ID NO:10; #201
  • EMSL3 SEQ ID NO:11; #301
  • EMSL5 D1 SEQ ID NO:13; #501
  • EMSL5 D10 SEQ ID NO:50; #510
  • EMSL8 SEQ ID NO:
  • FIG.4D shows unpublished X-ray structure of EMSL1 D1 (residues 1-268) in ribbon highlighting the disulfide bond in yellow; the carbohydrate binding sites (CBS#1 and #2) and conserved motifs (CXLXL; SEQ ID NO:1) are highlighted in red and yellow, respectively.
  • the structure is aligned with MSL2 (PDB ID: 5T20) represented in gray ribbon.
  • FIG.5 is a table showing how representative N-glycan TE motifs are classified by MotiFinder, including High mannose complex N-glycans, Asymmetric biantennary Complex N-glycans, Asymmetric biantennary Complex N-glycans with terminal fucosylation, Asymmetric biantennary Complex N-glycans with core fucosylation, and Symmetric Attorney Docket No.: 15024-372PC0 Patent biantennary Complex N-glycans. Glycans are rendered using the Symbolic Nomenclature for Glycans (SNFG).
  • FIG.6 is a graph showing surface lung tumor colonies in mice injected with line 66.1 cells after the indicated treatments with either TE or rMLS1.
  • FIG.7A shows the relative fluorescence units as result of TE binding to the glycan structures of the 100-glycan library (ZBiotech 100-glycan microarray).
  • FIG.7B shows the optimization of the glycan array conditions at different TE concentrations (explored concentrations 0.2 to 150.0 ⁇ g/mL).
  • FIG.7C also shows the calculation of an apparent KD for Man-5 and Man-6 glycans binding data to TE.
  • FIG.7D presents a representative slide showing fluorescence (light dots) of just high- mannose N-glycans. On this microarray, we can observe TE binding specificity to high- mannose N-glycans.
  • FIG.8A The concentration of 0.4 ⁇ g/mL fluorescently labeled TE (NHS-Alexa555- or AF555-TE) was assessed for N-glycan recognition pattern among 100 N-glycans (ZBiotech 100N-glycan library) in replicates of 8.
  • FIG.8B illustrates fluorescence of N-glycans on the printed 100 N-glycan microarray during the process of fluorescence detection.
  • FIG.8C shows glycan classification analyzed by MotifFinder.
  • FIG.9A through FIG.9E relate to TE inhibits experimental and spontaneous metastasis in two TNBC models.
  • FIG.9A and FIG.9B show data for counted lung tumor colonies by injection of either line 66.1 tumor cells or line 410.4 cells into syngeneic mice treated with either PBS or TE.
  • FIG.9C shows the effect of TE or PBS on spontaneous lung metastases from primary 410.4 tumors.
  • FIG.9D shows primary tumor growth in mice from treatment in FIG.9C.
  • FIG.9E is a Kaplan-Meier survival plot of mice injected i.v. with 66.1-luciferase cells and treated with TE or PBS.
  • FIG.10A shows the relative fluorescence units (RFU) (y-axis) of TE (blue bar) and MSL1 D34 (residues, 28-253) (red bar) on the 100 N-glycans array (x-axis).
  • REU relative fluorescence units
  • FIG.10B and FIG.10C illustrate the fluorescence of symmetric biantennary, asymmetric biantennary, and high-mannose N-glycans for TE (FIG.10B) and EMSL1 D34 Attorney Docket No.: 15024-372PC0 Patent (residues, 28-253) (FIG.10C) on the printed 100 N-glycan microarray library (ZBiotech TM 100N-glycan) during the process of fluorescence detection.
  • FIG.10C illustrate the fluorescence of symmetric biantennary, asymmetric biantennary, and high-mannose N-glycans for TE (FIG.10B) and EMSL1 D34 Attorney Docket No.: 15024-372PC0 Patent (residues, 28-253) (FIG.10C) on the printed 100 N-glycan microarray library (ZBiotech TM 100N-glycan) during the process of fluorescence detection.
  • FIG.11 examines binding of recombinant eGFP-EMSL1 (eGFP-EMSL1) in presence of unlabeled rEMSL1, rEMSL1 D1 -Y64A, or rMSL1 D1 -5xAla.
  • FIG.12A and FIG.12B show bioluminescent imaging in mice injected with 66.1- luciferase cells and treated with rEMSL1 D1 (SEQ ID NO:9; #001) or rEMSL1 D1 -Y64A.
  • rEMSL1 D1 SEQ ID NO:9; #001
  • rEMSL1 D1 -Y64A for FIG.12A, Balb/cByJ female mice treated daily with rEMSL1 (50 ⁇ g protein) or mutant Y64A protein for 10 days.
  • FIG 12A shows the top 15 ranked N-glycan structures identified for TE (blue) and EMSL1 D34 (residues, 28-253) (red) by 100 N-glycan microarray analysis.
  • FIG.13B shows a 100 N-glycan array for EMSL1 D34 CRS mutants, rEMSL1 D1 -Y64A (orange bar) and rEMSL1 D1 -5xAla (green bar), showing the absence of binding.
  • FIG 13C is a drawing showing representative N-glycan structures showing binding to rEMSL1 D34 : Complex type biantennary N-linked glycans as N001; Lewis X type N-glycans as in N224 (circled in red); Terminal Type 2 N-acetyllactosamine (LacNAc) as in N6030 (circled in blue).
  • FIG.14 presents competitive binding studies with line 66.1 cells and recombinant eGFP-EMSL1 (rEMSL1) (SEQ ID NO:27; #021) in the presence or absence of increasing concentrations of rEMSL1 (SEQ ID NO:9; #001).
  • FIG.15A provides data on inhibition of proliferation of murine 66.1 cells by rEMSL1 (SEQ ID NO:9; #001).
  • FIG.15B shows data on inhibition of proliferation of human breast cancer cell line MDA-MB-231 by the indicated treatments. As with TE, minor affects on proliferation were observed.
  • FIG.16 relates to the % inhibition of proliferation of 66.1 cells, comparing the effects of TE, rEMSL1 D1 (SEQ ID NO:9; #001), rEMSL1 D1 -Y64A (SEQ ID NO:#1001) and rEMSL1 D1 -5xAla (SEQ ID NO:#1001) versus vehicle-treated cells for the indicated compounds.
  • FIG.17 shows data on the migration of line 66.1 cells under the indicated treatments.
  • FIG.18 shows data for primary tumorsphere formation of 66.1 cells in response to TE or rEMSL1 D1 (SEQ ID NO:9; #001).
  • FIG.19 provides data on the effect of rEMSL1 D1 (SEQ ID NO:9; #001) on the fraction of aldehyde-dehydrogenase positive line 66.1 cells.
  • FIG.20A and FIG.20B presents a fluorescence microarray showing rEMSL1 D1 (SEQ ID NO:9;#001) (FIG.20A) and TE (FIG.20B) binding to PD-L1/PD-1.
  • FIG.21 is a table presenting a list of protein sequences engineered EMSL1-EMSL9 and ETCI1-ETCI3 based on proteomic analysis performed on TE samples and similarity searches using UniProt TM database (uniprot.org). DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 1. Overview [0056] This study has identified new therapies for breast cancer, including the most aggressive form, Triple Negative Breast Cancer (TNBC). Soluble proteins derived from an extract of the plant Colocasia esculenta, commonly known as taro, exhibit remarkably potent inhibitory effects on tumor metastasis in two orthotopic models of TNBC.
  • TNBC Triple Negative Breast Cancer
  • rEMSL1 engineered recombinant EMSL1 (rEMSL1) protein that replicates, with high specific activity (i.e. antimetastatic activity/mg protein), the anti-metastatic, anti-proliferative, anti- migratory and anti-stem cell activity of Taro Extract (TE).
  • This protein potently inhibits both metastatic and cancer stem cells (CSCs) with efficacy, in the absence of toxicities, and can be scaled up for commercial manufacture.
  • the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125 ⁇ 0.025, and “about 1.0” means 1.0 ⁇ 0.2. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein.
  • a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.
  • TE Trio Extract
  • TE contains a highly represented (>90% amino acid coverage) mannose specific lectin 2 (MSL2), previously named lectin (A5HMM7, GenBank Accession: EF541132.1) and mannose-specific lectin 1 (MSL1; SEQ ID NO:9), also known as taro storage protein TSP (Q39487, GenBank accession: D16173.1) from C. esculenta (>60 amino acid coverage).
  • MSL2 mannose specific lectin 2
  • SEQ ID NO:9 mannose-specific lectin 1
  • taro storage protein TSP Q39487, GenBank accession: D16173.1
  • Attorney Docket No.: 15024-372PC0 Patent Tarin or TAR1 (Q39418, GenBank accession: D16173.1) from C. esculenta also was identified with 45% amino acid sequence coverage.
  • rTSP or “rMSL1” and “rEMSL1” refers to recombinant proteins of the same name.
  • rTSP engineered mannose-specific lectin 1
  • TSP Taro Storage Protein
  • amino acid coverage means the percentage of amino acid sequences or peptides identified in TE sample analyzed by LC-MS/MS methods.
  • the term “treat,” “treatment,” and their cognates refer to administering a compound or pharmaceutical composition to obtain a desired pharmacologic and/or physiologic effect.
  • Treatment therefore includes: (a) preventing or reducing the likelihood of diagnosis with the condition or disease or symptoms thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; and/or (b) inhibiting, ameliorating, or reducing the condition or disease or symptom thereof, such as, arresting its development; and/or (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression of the condition or disease or symptom thereof; and/or (d) preventing or inhibiting metastasis, producing remission, diminishing of symptoms to make the disease, pathology or condition more tolerable to the patient, slowing the rate of degeneration or decline, or improving a patient's physical or mental well-being.
  • administering refers to introducing an agent to a subject, and can be performed using any of the various methods or delivery systems for administering agents, pharmaceutical compositions or delivering gene vectors known to those skilled in the art. Common methods of administering agents include oral, intravenous injection or infusion, transdermal, transmucosal, intraperitoneal, intratumoral, and the like.
  • therapeutic and its cognates refer to an action taken with the intention of benefitting a patient and may comprise administration of a chemotherapeutic agent such as a drug, antibody, or the like. Therapies also include radiation therapy, surgery, dietary regimens, and the like as well.
  • a therapeutically effective amount refers to an amount that produces a desired pharmacologic or physiologic response in a patient.
  • the therapeutically effect amount can refer to a single dose or a number of divided doses or to a dosage regimen that can last one day, several days, weeks, months, or indefinitely. The therapeutic effect may occur immediately after administration of the amount or be delayed for hours or for a considerable time.
  • a therapeutically effective amount for an adult human includes amounts from about 1 ⁇ g/kg/day to about 50 mg/kg/day of purified protein.
  • the term “subject,” “individual,” “host,” and “patient,” generally are used interchangeably to refer to any animal suitable for treatment by the inventive compounds, compositions, or methods.
  • Subjects preferably are humans, primates, simians, rodents and the like, but also include any mammal such as farm animals, companion animals, service animals, laboratory animals, and the like.
  • a “subject in need” refers to a subject that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be treated or prevented by administration of compounds and/or compositions described as embodiments of the invention here.
  • a disease or condition is a hyperproliferative disease or disorder.
  • hypoproliferative disease or disorder refers to a benign or malignant condition, involving hyperproliferation of a cell population or tissue, including cancers, as discussed below.
  • pharmaceutical composition refers to a composition that comprises an active agent, preferably rEMSL1 or a pro-drug thereof, in combination with a pharmaceutically acceptable carrier or excipient.
  • a recombinant protein has been produced in bacteria that partially or fully replicates activity we identified from an extract produced from the natural Taro plant. Active components of this taro extract (TE) were identified as two 12-kDa degradation products of the taro storage protein that we engineered for overexpression (termed EMSL1). A new Attorney Docket No.: 15024-372PC0 Patent recombinant protein, termed rEMSL1, was produced to replicate inhibitory components of TE on metastasis, proliferation, migration and CSCs. This protein can be useful for treatment of hyperproliferative disorders.
  • Taro-derived proteins such as those in TE, can effectively target metastatic tumor cells and can inhibit CSC by direct binding to glycan ligands known to be aberrantly expressed in multiple cancers.
  • the TE and rEMSL1 compounds produce a blockade of glycan-dependent receptor-mediated processes, which are required for metastasis.
  • the engineered rEMSL1 compounds and compositions of certain embodiments of the invention are designed to have higher efficacy and bioavailability.
  • Plant materials which can provide a source of the antimetastatic or anticyclooxygenase activity useful in embodiments of the invention preferably are members of the genus Colocasia, commonly referred to as taro, eddoe and dasheen.
  • Suitable species such as, for example, Colocasia affinis, C. bicolor, C. esculenta, C. fallax, C. fontanesii, C. formosan, C.a gaoligongensis, C. gigantean, C. gongii, C. gracilis, C. heterochroma, C. humilis, C. konishii, C. latifolia, C. lihengiae, C.
  • members of the genus Xanthosoma also can be used as a source for the compositions.
  • plants commonly referred to as Yautia, Malanga Blanca, Tannia, Cocoyam, Eddo, Coco, Sato-imo, Japanese Potato, Macabo, Taioba, Dasheen, Quequisque, Ape and Tannier can be used.
  • plants belonging to species including, for example, Xanthosoma sagittifolium, X. atrovirens, X. violaceum, X. maffaffa, X. weeksii, X. roseum, X. daguense, X. poeppigii, X.a hastifolia, X. robusturn, X. caracu, X. wendlandii, X. pichinchense, X. hannoniae, X. nigrum, X. lindenii, X.a narinoense, X. eggersii, and X. yucatanensis are suitable for use. 2.
  • TE is an aqueous extract of a plant, including Colocasia and Xanthosoma spp., preferably the “taro” plant Colocasia esculenta.
  • the extract or isolated polypeptides contained in the extract are made by a process comprising obtaining the Attorney Docket No.: 15024-372PC0 Patent uncooked corm from the root (such as Taro, Malanga or Yautia), peeling the corm, combining it with an aqueous solution (such as PBS); blending the corm to liquefy it, centrifuging this liquid (e.g., at 1200 rpm for 15 minutes at 4°C) the liquid to obtain a supernatant, centrifuging the supernatant at high speed (e.g., at 15,000 rpm for 20 minutes at 4°C), and filter sterilizing the resulting supernatant to obtain a stock water-soluble extract.
  • the stock water-soluble extract is adjusted to have a protein concentration of about 1-5 mg/mL.
  • the stock water-soluble extract can be further purified by various techniques that are known in the art, including centrifugation, size exclusion chromatography, ion exchange chromatography, reversed phase liquid chromatography, reversed phase high performance liquid chromatography, and/or a combination of these approaches, to yield a more purified or substantially pure active agent.
  • the stock extract can be centrifuged through molecular weight limit devices (such as, for example, Amicon TM Ultra 10 K (10,000) Nominal Molecular Weight Limit (NMWL) devices (Millipore TM Corporation) at 4000 g for 45 minutes at 25°C).
  • the upper fraction, which contains the antimetastatic activity can be filter sterilized (e.g., using a 0.2 micron filter).
  • the stock extract can be further purified using size exclusion chromatography (SEC).
  • preparative SEC can be performed on a Biosuite TM 250, 13 micron, 21.5 x 300 mm column (Waters TM Corp.) using Dulbecco's phosphate buffered saline with calcium and magnesium, at a flow rate of 2 ml/min. Fractions can be collected every 30 seconds and tubes can be pooled based on UV absorbance at 220 nm. In this system, the antimetastatic activity resides in an approximately 30 kD fraction (calibrated using BSA and carbonic anhydrase globular protein). [0082] In some embodiments, the 30 kD fraction from size exclusion chromatography can be further purified by ion exchange chromatography.
  • RPLC reversed phase chromatography
  • Analytical RPLC can be done using standard techniques and equipment (e.g., using a Jupiter TM C5300 ⁇ column (Phenomenex TM ), employing a 40-minute gradient of 1-100% B at 1 ml/min.
  • Buffer A 0.1% trifluoroacetic acid (TFA) in water;
  • Buffer B 0.1% TFA in water:acetonitrile (20:80), with UV detection at 215 nm using a Beckman Coulter TM HPLC systems with System Gold V8 or 32 Karat software packages).
  • additional purification of isolated proteins can be accomplished using reversed phase high performance liquid chromatography using standard techniques and equipment (e.g., on a Waters TM 2695 HPLC system; absorbance can be monitored with an Applied Biosystems TM 785 UV detector at 214 nm; proteins can be separated, e.g., on a Waters TM Symmetry 3003 micron C41 mm x 150 mm column with a gradient of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.09% TFA in acetonitrile (solvent B)).
  • TFA trifluoroacetic acid
  • EMSL1 Engineered Mannose-Specific Lectin 1
  • MSL1 mannose-specific lectin 1
  • TSP taro storage protein TSP (Q39487, GenBank accession: D16173.1) from C. esculenta (>60 amino acid coverage).
  • the sequence of EMSL1 (also known as 12kD Storage) is given below. Peptides in bold are unique to each sequence.
  • TE contains highly represented (>90% amino acid coverage) mannose specific lectin 2 (MSL2), also known as lectin (A5HMM7, GenBank Accession: EF541132.1).
  • MSL3 mannose-specific lectin 3
  • MAKLLLFLLPAILGLLIPRSAVALGTNYLLSGQTLNTDGHLKNGD FDLVMQNDCNLVLYNGNWQSNTANNGRDCKLTLTDYGELVIK NGDGSTVWRSRAKSVKGNYAAVLHPDGRLVVFGPSVFKIDPWV PGLRFRNIPFTDNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLV LYGGKYGWQSNTHGNGEHCFLRLNHKGELIIKDDDFKTIWSSN SSSKQGDYVLILRDDGFAVIYGPAIWETSA (MSL3; SEQ ID NO:11) [0088] Surprisingly, two variants of tuber agglutinin CEA from C.
  • CEA1 R9RL27, NCBI accession: JX435122.1
  • CEA2 also known as hypothetical taro protein (NCBI accession: MQM11449.1) from C. esculenta (see Table 1, below).
  • CEA2 is a variant of CEA1 (24-252) - S164N/D209E/S217R.
  • these two lectins are formed by unique peptides that can be unequivocally assigned to each one of them, i.e.
  • CEA1 MAKLLLFLLPAILGLLVPRSAVALGTNYLLSGQTLDREGHLKNG DFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDYGELVI KNGDGSTVWRSRAQSVKGNYAAVVHPDGRLVVFGPSVFKIDP WVPGLNSLRFRNIPFTNNLLFSGQVLYGDGRLTAKSHQLVMQG DCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKGELIIKDDDFKT IWSSSSSSKHGDYVLILRDDGFAVIYGPAIWETSPQAK (SEQ ID NO:69)
  • CEA2 MAKLLLFLLPAILGLLVPRSAVALGTNYLLSGQTLDREGHLKNG DFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDYGELVI KNGDGSTVWRSRAQSVKGNYAAVVHPDGRLVVFGPSVFKIDP WVPGLNSLRFRNIPFTNNLLFSGQVLYGDGRLTAKNHQLVMQG DCNLV
  • esculenta was also performed against TE homologue lectins with similar biophysical properties, i.e., curculin, miraculin, sporamin, and Kunitz trypsin inhibitors.
  • Two 25 kDa storage proteins were recognized, called inhibitors of trypsin/chymotrypsin ITC1 (GenBank accession: D16174.1; also known as trypsin/chymotrypsin inhibitor or ECT1) and ITC2 (GenBank accession: D16175.1; also known as ECT2), with 80% and 70% amino acid coverage, respectively, and assigned the isolated peptides with high confidence (see Table 1 and Table 2). All together, we report here the most comprehensive characterization of isolectins in TE from C. esculenta.
  • ETCI1 MEFVLLLVSSLLLTARAAVASNPVLDVDGNELRRGNRYYAISL RSPNSGLTLAVRSNAPCPLNVDQAPSKDYGRPLAFFPENVDDD TVQEGSTLYIMFPEPSECRESTVWTLGRETDVVTTGGTSSSAIG PRNSRFTIRRTGDASSKGEYQIEVCPCSIGVSRAPCRLACVGSLG LTEDEANLLLNINNERPHTVRFVKVKEELAASRR (SEQ ID NO:71)
  • ETCI2 MEFILLLVSSLLLTARAAAASNPILDVDGDELRRGHRYYAISER RPVSGLTLAARSNAPCPLNVAQTSSNDYGRPLAFFPENAEDD TVQEGNTLNIMFPEPTECRASTVWTLDRERGVVTTGGTSSSA IGPHNSRFSIRRAGDASSERERKYQIEVCPCSNGVPRPSCRMAC VGSLGLTEDEGNLLLNINNERPHAIRFVKVKEELPA
  • rEMSL1 Recombinant EMSL1
  • rEMSL1 is produced by recombinant means, from a bacterium, such as, e.g. E. coli or any suitable microorganism. See below for the sequences used for production (Table 3).
  • the invention relates to a method for producing rEMSL1 in HEK293 cells as herein described.
  • rEMSL1 like TE, possesses high binding affinity to specific glycan motifs including Lewis X type carbohydrates, type 2 N-AcetylLactosamine (LacNAc) and core fucosylated N- glycans including the glycan motifs detected in HER-2+ or TNBC and paired metastatic specimens from the same individuals. Branched and fucosylated N-glycans (Man2- 9/HesNAc2/Fuc0-1) have been observed in primary and paired metastatic tissues, which proposed to serve as prognostic markers for patients. Like TE, rEMSL1 potently inhibits metastasis in two syngeneic models of TNBC. See Examples below.
  • rEMSL1 like TE, shares potent anti-metastatic activity with TE in that rEMSL1 inhibits proliferation of murine and human breast cancer cells as well as tumor cell migration. Breast cancer stem cells are sensitive to rEMSL1-mediated inhibition. See Examples below. 5. Methods [0095]
  • LC-MS/MS spectra of tryptic digest of proteins from TE sample were searched against the fasta sequences from uniprot database for Colocasia esculenta using Attorney Docket No.: 15024-372PC0 Patent Byonic TM software and manually with trypsin and/or chymotrypsin as digestion enzyme with specific cleavage option enabled.
  • TSP taro storage protein
  • MSL1 mannose specific lectin 1
  • GNA Galanthus nivalis agglutinin
  • GNA Lectins bind glycans expressed on cell membranes. Aberrant glycosylation is associated with higher metastatic potential in breast and other malignancies.
  • Bacterial lysates were prepared in buffer containing 50 mM Tris-HCl (pH 8), 500 mM NaCl, 8 M Urea, 5 mM beta-mercaptoethanol (BME), 0.5 mM AEBSF, DNase solution, 10 mM MgCl 2 , 10 mM EDTA and sonicated. Cell debris were removed by centrifugation at 15,000 x g for 45 minutes at 4 °C.
  • the sample was filtered and loaded onto a previously equilibrated HiPrep 16/60 IMAC column with buffer A (15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME) and B (buffer A plus 1 M Imidazol).
  • buffer A 15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME
  • B buffer A plus 1 M Imidazol
  • Sumo-fused rEMSL1 eluted fractions (molecular weight ⁇ 45kDa) were pooled and refolded on column decreasing urea concentration step- wise using buffer 15 mM Tris-HCl (pH 8), 500 mM NaCl, 5 mM BME, 1% Glycerol.
  • Attorney Docket No.: 15024-372PC0 Patent [0100] Cleavage of the Sumo fusion protein was performed at 4 °C by addition of 150 ⁇ L of 1mM Ulp1 protease to refolded Sumo-MSL1 protein. The totality of the cleavage reaction was checked by SDS-PAGE.
  • a second HiPrep 16/60 IMAC column was performed to remove the His-tagged Sumo protein and the His-tagged Ulp1 protease.
  • MSL was collected in the flowthrough (>95% purity) as expected and verified by SDS-PAGE.
  • rMSL was concentrated and injected onto a Superdex TM S200-PG size exclusion column previously equilibrated with buffer 10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM TCEP. Fractions containing MSL (>99% pure) were eluted from the S200 column (fractions 13-15) and identified using SDS-PAGE.
  • a size exclusion column S200 was calibrated with a gel filtration standard kit (Biorad TM ), containing thyroglobulin, bovine ⁇ -globulin, chicken ovalbumin, equine myoglobin, and vitamin B12, with M.W. 670,000; 158,000; 44,000; 17,000, and 1,350 g/mol, respectively.
  • Therapeutic Agents [0102] The invention, in certain embodiments, provides the following compounds, which can be used for treatment of hyperproliferative diseases and conditions, including especially cancer.
  • Embodiments of the invention include fusion constructs, including cloning the active components of TE into pcDNA 3.1(-) Mammalian Expression Vector) as linear fusion proteins: a.
  • Embodiments of the invention include fusion constructs, including cloning the active components of TE into pcDNA 3.1(-) Mammalian Expression Vector) as linear fusion proteins in combination with ETCI proteins: Attorney Docket No.: 15024-372PC0 Patent a.
  • Embodiments of the invention include fusion constructs, including cloning the active components of TE into pcDNA 3.1(-) Mammalian Expression Vector) as a trimerization fusion protein: a.
  • the invention also includes certain embodiments wherein the therapeutic agent is administered in the form of a prodrug. 7.
  • Pharmaceutical Compositions Any of the therapeutic agents discussed herein can be used to produce a pharmaceutical composition according to embodiments of the invention.
  • the pharmaceutical compositions can include one or more of the peptide or recombinant peptide compounds disclosed herein.
  • the pharmaceutical compositions can include one or more inventive compound and also and further comprise one or more additional therapeutic agent known in the art, which can be determined by the skilled practitioner according to standards of care.
  • the compounds can be administered as a base compound, and any pharmaceutically acceptable hydrate, solvate, acid or salt, and can be amorphous or in any crystalline form, or as an oil or wax.
  • any pharmaceutically acceptable salt can be used, as may be convenient.
  • the compounds described herein are formulated and are administered as a pharmaceutical composition that includes a pharmaceutically acceptable carrier and one or more pharmaceutical/therapeutic agent, including one or more of the compounds described herein, and including one or more of the inventive compounds described herein with an additional agent, such as drug of another class.
  • a pharmaceutically acceptable carrier refers to any convenient compound or group Attorney Docket No.: 15024-372PC0 Patent of compounds that is not toxic and that does not destroy or significantly diminish the pharmacological activity of the therapeutic agent with which it is formulated.
  • Such pharmaceutically acceptable carriers or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers or excipients known in the art.
  • a suitable carrier depends on the route of administration contemplated for the pharmaceutical composition.
  • Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated. Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated, including local or systemic administration.
  • routes of administration can include, but are not limited to local or parenteral routes, including: oral, intravenous, intratumoral, intraarterial, intrathecal, intramuscular, subcutaneous, intradermal, intraperitoneal, rectal, vaginal, topical, nasal, local injection, buccal, transdermal, sublingual, inhalation, transmucosal, wound covering, direct injection into an area to be treated, and the like.
  • the administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art.
  • the forms which the pharmaceutical composition can take will include, but are not limited to: tablets, capsules, caplets, lozenges, dragees, pills, granules, powders, oral solutions, powders or granules for dilution in a suitable solvent, powders for inhalation, vapors, gases, sterile solutions or other liquids for injection or infusion, transdermal patches, buccal patches, inserts and implants, rectal suppositories, vaginal suppositories, creams, lotions, oils, ointments, topical coverings (e.g., wound coverings and bandages), suspensions, emulsions, lipid vesicles, and the like.
  • Treatment regimens of the chemical compounds contemplated for use with the invention include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including indefinitely or administration for the remainder of the subject’s life.
  • the regimen can include multiple doses per day, one dose per day or per week, for example, or one or more long infusion administration lasting for an hour, multiple hours, a full day, or longer.
  • Suitable subjects for the invention include mammalian laboratory animals, mammalian farm animals, mammalian sport animals, mammalian zoo animals, and mammalian companion animals.
  • the subject is a human, but subjects can include mammals such as simians, felines, canines, equines, rats, mice, rabbits, bovines, porcines, ovines, caprines and the like.
  • Modes of administering include, but are not limited to, any of those discussed above with respect to routes of administration.
  • Administration also can refer to introducing a nucleic acid construct to the subject as DNA or mRNA, introducing a vector containing the nucleic acid to the subject, or introducing cells that have been transduced ex vivo with a construct, such as by electroporation or using a vector as described herein, to the subject.
  • Dosage amounts per administration of these compounds include any amount determined by the practitioner and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the condition to be treated, the severity of the condition, and the like.
  • a dose in the range of about 0.01 mg/kg to about 100 mg/kg is suitable, preferably about 0.1 mg/kg to about 50 mg/kg, more preferably about 0.1 mg/kg to about 10 mg/kg, and most preferably about 0.2 mg/kg to about 5 mg/kg are useful.
  • This dose can be administered weekly, daily, or multiple times per day.
  • a dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered per dose or per day.
  • the compositions of the invention can be administered in combination with (concurrently or sequentially with) existing cancer therapies.
  • Suitable conditions or diseases for treatment using the invention include hyperproliferative diseases or conditions, including ductal carcinoma in situ, but preferably one or more cancer.
  • Suitable cancers which can be treated using the compositions and methods of the present invention include cancer of the oral cavity and pharynx (lip, tongue, salivary gland, floor of mouth, gum and other mouth, nasopharynx, tonsil, oropharynx, hypopharynx, other oral/pharynx; i.e.
  • cancers of the digestive system esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; liver; intrahepatic bile duct; gallbladder; other biliary; pancreas; retroperitoneum; peritoneum, omentum, and mesentery; other digestive
  • cancers of the respiratory system nasal cavity, middle ear, and sinuses; larynx; lung and bronchus; pleura; trachea, mediastinum, and other respiratory
  • cancers of the mesothelioma bones and joints; and soft tissue, including heart; skin cancers, including melanomas and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer
  • cancer of the female genital system cervix uteri; corpus uteri; uterus, nos; ovary; vagina; vulva; and other female genital
  • Neoplasm malignant
  • Carcinoma NOS
  • Carcinoma undifferentiated, NOS
  • Giant and spindle cell carcinoma Small cell carcinoma, NOS; Papillary carcinoma, NOS; Squamous cell carcinoma, NOS; Lymphoepithelial carcinoma; Basal cell carcinoma, NOS; Pilomatrix carcinoma; Transitional cell carcinoma, NOS; Papillary transitional cell carcinoma; Adenocarcinoma, NOS; Gastrinoma, malignant; Cholangiocarcinoma; Hepatocellular carcinoma, NOS; Combined hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyp; Adenocarcinoma, familial polyposis coli; Solid carcinoma, NOS; Carcinoid tumor, malignant; Branchiolo-alveolar
  • breast cancer such as TNBC, HER-2+ and HR+ breast cancer
  • Metastatic malignancies are particularly contemplated for use according to embodiments of the invention, including metastasis to the brain or other organs such as lung, liver, bone, and the like.
  • the peptides disclosed here are contemplated for use in treatment of subjects for the conditions described and listed above.
  • Particular preferred peptides include, but are not limited to SEQ ID NOs:9, 10, 11, 12, 13, 16, 71, 72 and 74-113. 9.
  • rMSL 1-269 Based on examining data from our isolation and characterization of TE we engineered and claim a sequence, termed rMSL 1-269 , that recapitulates a large component of the lectin binding specificity identified in TE. [0122] Based on data from our laboratory, we identified bacterial expression systems, which could overproduce the rMSL 1-269 sequence within multiple expression systems, one of which was superior (list here) and enabled us to produce high-yields (mg/liter) of the active and highly stable rMSL 1-269 protein, which was fully sufficient for biophysical characterization and future engineering using standard methods.
  • Standard screening methods will be completed to determine the optimal construct and mammalian cell line, as well as via stable or transfection strategies, as is standard in the field for optimizing production, purification, and manufacturing of an isolated biologic containing a single composition of matter identity, such as rMSL 1-269 .
  • Some of these additional testing steps on product 2 can involve systematic removal of each rMSL component of the mixture, to identify which, if any of the rMSL# sequences can be removed from the mixture, such that improved specific activity values of the resulting product are obtained.
  • a detailed engineering of Attorney Docket No.: 15024-372PC0 Patent scale-up and biomanufacturing can be achieved for product 4 using standard methods in the field.
  • Product 5 - Next standard methods can be used to determine an optimal ratio for the remaining components of the mixture. This can be achieved via systematic screening of mixture ratios with standard methods established here, on a small scale.
  • Mass Spectroscopy [0131] Mass Spectroscopy. [0132] Mass spectrometry reagents were purchased from Sigma Aldrich TM unless otherwise mentioned. Sequencing-grade modified trypsin was purchased from Promega TM . Mass spectrometric data acquisition was performed on a Thermo Scientific TM LTQ Orbitrap TM Fusion Tribrid mass spectrometer attached with a Dionex TM nano-LC system and on AB SCIEX MALDI TOF/TOF 5800 (Applied Biosystem TM MDS Analytical Technologies) mass spectrometer. Data analysis was performed by using Data Explorer V4.5, Xcalibur 3.0, Byonic TM software and Glycoworkbench 1.1. [0133] B.
  • SDS-PAGE bands from TE sample were cut into 1 mm 2 pieces and destained by adding 100 ⁇ L acetonitrile: NH4HCO3 (1:1) (ACN:50mM AMBIC BUFFER) and incubated at room temperature (RT) for 30 minutes. Tubes were centrifuged at 14,000 rpm, the supernatant was discarded, and gel pieces were resuspended in 100 ⁇ L of ACN. Tubes were incubated for another 20-30 minutes and centrifuged. This process was repeated three times.
  • the proteins in gels then were reduced by adding 50 ⁇ L of DTT solution (25 mM), carbamidomethylated by adding 50 ⁇ L of iodoacetamide solution (25 mM) and finally washed with 200 ⁇ L acetonitrile.
  • Fifty microliters of digestion buffer was added to the gel pieces and proteins were digested by adding 5 ⁇ L of sequencing-grade trypsin and/or chymotrypsin (Promega TM ) and incubated at 37 oC for 12 hours.
  • the digested peptides were extracted in 5% formic acid in 1:2 water-acetonitrile.
  • the precursor ion scan was acquired at 120000 resolution in an Orbitrap TM analyzer and precursors at a time frame of 3 seconds were selected for subsequent fragmentation using either an HCD product triggered CID program.
  • the threshold for triggering an MS/MS event on an ion-trap was set to 500 counts.
  • Charge state screening was enabled, and precursors with unknown charge state or a charge state of +1 were excluded (positive ion mode).
  • Dynamic exclusion was enabled (exclusion duration of 30 seconds).
  • the fragment ions were analyzed on the Orbitrap TM equipments for HCD and CID at 30000 resolution. [0137] Analysis of proteins and glycoproteins was performed as follows.
  • Peptides in green color font are detected after trypsin digest, and peptides in blue color font are detected after trypsin & chymotrypsin digests. Overlapped of these two peptides is possible and is colored in blue also. Underlined peptides highlight unique peptides to each protein. [0139] Other known data for the sequences derived from Byonic TM and manual inspection are also presented. Carbamidomethylation as fixed modification, oxidation of methionine as variable modification, was used as search parameters. The LC-MS/MS spectra were also analyzed manually for the glycopeptides with the support of Xcalibur TM software.
  • the HCD and CID MS2 spectra of glycopeptides were evaluated for the glycan neutral loss pattern, oxonium ions and the glycopeptide fragmentations to assign the sequence and the presence of glycans in the glycopeptides.
  • Table 1 List of peptides identified by searched against the fasta sequences from UniProt database using Byonic TM software and manually with trypsin and/or chymotrypsin as digestion enzyme with specific cleavage option enabled.
  • Residues LRFR corresponds to a common cleavage site. Peptides in green color font are detected after trypsin digest, and peptides in blue color font are detected after trypsin & chymotrypsin digests. Overlapped of these two peptides is possible and is colored in blue also. Underlined peptides highlight unique peptides to each protein.
  • the HCD and CID MS 2 spectra of glycopeptides were evaluated for the glycan neutral loss pattern, oxonium ions and the glycopeptide fragmentations to assign the sequence and the presence of glycans in the glycopeptides.
  • the glycans were dried with nitrogen gas and profiled by MALDI-TOF and ESI-MS.Enzymatic Release of N-glycans.
  • the extracted glycopeptides of each TE band (12 kDa, 23 kDa, and 52 kDa) sample were taken for N-linked glycan profiling.
  • the treatment of PNGase F and/or PNGAse A at 37oC for 16 hours was used to cleave the N-glycans.
  • the glycans were purified with a C18 cartridge, permethylated, and then analyzed with MALDI-TOF-MS.
  • the sample in 5 % acetic acid ( ⁇ 3 mL) was passed through a C18 cartridge and collected the flow through containing released glycans.
  • the cartridge is washed with 2 mL of 5 % acetic acid and the collected fractions were dried by lyophilization.
  • the released N-linked glycans were permethylated for the structural characterization by mass spectrometry.
  • Permethylation of glycans was performed as follows. The glycans were permethylated for structural characterization by mass spectrometry using previously reported protocol. Briefly, the dried eluate was dissolved with dimethyl sulfoxide and methylated by using methyl iodide on DMSO/NaOH mixture.
  • the slide was analyzed in a fluorescence scanner and the data reported as relative fluorescence units (RFU), corrected by RFU of negative control, for each glycan structure identified by a number on the x-axis.
  • the samples were analyzed on the glycan arrays provided by Z Biotech TM at indicated concentrations.
  • the assay arrays were read out by a microarray scanner (Innopsys Innoscan TM 710). Symbolic glycan structures were drawn using DrawGlycan-SNFG which uses Symbol Nomenclature for Glycans (SNFG). Analysis of the relationship of glycan motifs and the amount of TE binding was performed using MotifFinder TM program. [0146] D.
  • Bacterial lysates were prepared in buffer containing 50 mM Tris-HCl (pH 8), 500 mM NaCl, 8 M Urea, 5 mM beta-mercaptoethanol (BME), 0.5 mM AEBSF, DNase solution, 10 mM MgCl2, 10 mM EDTA and sonicated. Cell debris were removed by centrifugation at 15000 xg for 45 minutes at 4 °C. The sample was filtered and loaded onto a previously equilibrated HiPrep TM 16/60 IMAC column with buffer A (15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME) and B (buffer A plus 1 M imidazol).
  • Sumo-fused EMSL1 was eluted at 200 ⁇ 20 mM imidazole and analyzed by SDS-PAGE. Sumo-fused EMSL1 eluted fractions (molecular weight ⁇ 45kDa) were pooled and refolded Attorney Docket No.: 15024-372PC0 Patent on column decreasing urea concentration step-wise using buffer (15 mM Tris-HCl (pH 8), 500 mM NaCl, 5 mM BME, 1% Glycerol). [0148] Cleavage of the Sumo fusion protein was performed at 4 °C by addition of 150 ⁇ L of 1mM Ulp1 protease to refolded Sumo- EMSL1 protein.
  • EMSL1 was collected in the flowthrough (>95% purity) as it was expected and verified by SDS-PAGE. As a final purification step, EMSL1 was concentrated and injected onto a Superdex TM S200-PG size exclusion column previously equilibrated with buffer 10 mM Tris- HCl, pH 7.0, 150 mM NaCl, 1 mM TCEP.
  • Size exclusion column S200 was calibrated with a gel filtration standard kit (Biorad #1511901, Hercules, CA), containing thyroglobulin, bovine ⁇ -globulin, chicken ovalbumin, equine myoglobin, and vitamin B12, with M.W.670,000; 158,000; 44,000; 17,000, and 1,350 g/mol, respectively.
  • Crystals were obtained in 0.1M MES pH 5.0, 10% PEG 6000 and diffracted at 3.7 ⁇ resolution at a synchrotron radiation source.
  • TE and rEMSL1 were detected by incubation with cyanine3-labeled streptavidin at 0.5 mg/ml under a cover slip in the same buffer for 1 hour at room temperature.
  • the slide was analyzed in a fluorescence scanner and the data reported as relative fluorescence units (RFU), corrected by RFU of negative control, for each glycan structure identified by a number on the x-axis.
  • the samples were analyzed on the glycan arrays provided by Z Biotech TM at the indicated Attorney Docket No.: 15024-372PC0 Patent concentrations.
  • the assay arrays were read out by a microarray scanner (Innopsys Innoscan TM 710). Analysis of binding data was performed by Prism TM .
  • F. Mice [0153] Syngeneic Balb/cByJ or Balb/c/SCID female mice were purchased from Jackson Laboratories TM . All mice were housed in microisolator cages, fed conventional, autoclaved chow and provided drinking water ad libitum. [0154] G. Taro Extract. [0155] Taro extract (TE) was prepared as previously described in United States Patent No. 8,865,642, Example 1, which is hereby incorporated by reference, from peeled, homogenized corm of Colocasia esculenta. Briefly, commercially obtained Taro corm was peeled, combined with PBS in a weight:volume ratio of 1:3, and blended at low speed, followed by high speed to liquefy.
  • TE Taro extract
  • Murine mammary tumor cell lines (66.1, 66.1-luciferase and 410.4) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products TM , Inc.), 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 ⁇ g/ml) and 0.1 mM nonessential amino acids in a 10% and 5% CO2 humidified atmosphere, respectively.
  • FBS fetal bovine serum
  • penicillin 100 units/ml
  • streptomycin 100 ⁇ g/ml
  • 0.1 mM nonessential amino acids in a 10% and 5% CO2 humidified atmosphere, respectively.
  • Human breast cancer cell line MDA-MB-231 was maintained in DMEM supplemented with 10% FBS, 2mM glutamine, penicillin, streptomycin in a 10% CO2 humidified atmosphere.
  • Murine cell lines have been maintained in the laboratory for more than 30 years. The human cell line was acquired from collaborators and all cell lines have been verified within the last 5 years.
  • For cell viability assays cells were seeded in 24-well plates and PBS, rEMSL1 or TE Attorney Docket No.: 15024-372PC0 Patent was added at time 0. Seventy-two hours later, cell metabolic activity, as an indicator of cell growth, was determined by MTT assay following the manufacturer’s instructions (Sigma TM Chem. Co.). [0159] I.
  • GFP-rMSL1 binding assay [0160] Tumor cell lines were incubated with recombinant eGFP-EMSL1, unlabeled rEMSL1, rEMSL1-Y64A or rEMSL1-5xAla. After incubation, cells were centrifuged and washed two times and fluorescence intensity was analyzed by FACSCanto II cytometer and data analyzed with FlowJo software. [0161] J. Metastasis assay. [0162] The metastasis assay was conducted according to known methods. Briefly, PBS, TE or rEMSL1 was injected intraperitoneally in a volume of 200 ⁇ L, into syngeneic Balb/cByJ female mice on days 1-4.
  • 1-2 x 10 5 line 66.1 or 410.4 tumor cells were injected into the lateral tail vein. Treatment continued daily for an additional 6 days. Between days 14 -21 post tumor cell injection, when control animals became moribund, mice were euthanized, and surface lung tumor colonies were counted under a dissecting microscope. [0163] K. Migration assay. [0164] Tumor cells were suspended in OPTI-MEM medium and placed in the upper well of Millicell TM tissue culture (24 well) plate well inserts, 8 or 12 ⁇ m (Millipore TM ).
  • FIG.2A and FIG.2B show TE proteins resolved in SDS-PAGE (4-20%) on mainly 3 bands corresponding to 12, 23 and 52 kDa.
  • Ten microliters of whole TE sample (1mg/ml) was aliquoted, evaporated to 2 ⁇ L under stream of nitrogen and added to a matrix of sinapinic acid (SA, 20 mg/mL in 50% v/v methanol:water).
  • BSA (1 mg/ml) was used as positive control for MALDI-TOF analysis. The extrapolation of the molecular weight under the peak is provided and calculated after calibration of the S200 column with standards.
  • MSL4 Coverage by MS analysis The analysis covered 60% of 5J76 sequence .
  • MSL5 Coverage by MS analysis The analysis covered 81% of R9RL27 sequence .
  • ASNPVLDVDGNELRRGNRYYAISLRSPNSGLTLAVRSNAPCPLNVDQA Attorney Docket No.: 15024-372PC0 Patent VGSLGLTEDEANLLLNINNE RPHTVRFVKVKEELAASRR (SEQ ID NO:71) Total: 186; Red: 91; Coverage: 49%. 8. ETCI2 Coverage by MS analysis. The analysis covered 74% of Q39489 sequence.
  • EMSL1 28 LGTNYLLSGQTLETEGHLKNGDFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDHGE 87 A5HMM7v 24 LGTNYLLSGQTLNTDGHLKNGDFDLVMQNDCNLVLYNGNWQSNTANNGRDCKLTLTDYGE 83 EMSL1 88 LVINNGDGSTVWRSGAQSVKGDYAAVVHPEGRLVVFSPSVFKIDPSVPGLNSLRFRNIPF 147 A5HMM7v 84 LVIKNGDGSTVWRSRAKSVKGNYAAVLHPDGRLVVFGPSVFKIDPWVPGL------NIPF 143 EMSL1 148 TNNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 207 A5HMM7v 144 TDNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 203
  • EMSL1 28 LGTNYLLSGQTLETEGHLKNGDFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDHGE 87 R9RL2724 LGTNYLLSGQTLDREGHLKNGDFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDYGE 83 EMSL1 88 LVINNGDGSTVWRSGAQSVKGDYAAVVHPEGRLVVFSPSVFKIDPSVPGLNSLRFRNIPF 147 R9RL2784 LVIKNGDGSTVWRSRAQSVKGNYAAVVHPDGRLVVFGPSVFKIDPWVPGLNSLRFRNIPF 143 EMSL1 148 TNNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 207 R9RL27144 TNNLLFSGQVLYGDGRLTAKSHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 203 EMSL1 208
  • EMSL1 28 LGTNYLLSGQTLETEGHLKNGDFDLVMQDDCNLVLYNGNWQSNTANKGRDCKLTLTDHGE 87 B5LYJ924 LGTNYLLSGQTLDTEGHLKNGDFDLVMQDDCNLVLYNGNWQSNTANNGRDCKLTLTDYGE 83 EMSL1 88 LVINNGDGSTVWRSGAQSVKGDYAAVVHPEGRLVVFSPSVFKIDPSVPGLNSLRFRNIPF 147 B5LYJ984 LVIKNGDGSTVWKSGAQSVKGNYAAVVHPDGRLVVFGPSVFKIDPWVPGLNSLRFRNIPF 143 EMSL1 148 TNNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 207 B5LYJ9144 TNNLLFSGQVLYGDGRLTAKNHQLVMQGDCNLVLYGGKYGWQSNTHGNGEHCFLRLNHKG 207 B5LY
  • FIG.2C contains an intact mass spectrum of a TE sample by MS-ESI.
  • Example 3 MALDI-TOF-MS.
  • Extracted glycopeptides of TE bands (12kD, 23kD, and 52 kD) were obtained by extraction of the TE resolved band in SDS-PAGE (FIG.2B). Samples were taken for N- linked glycan profiling.
  • Example 4 Protein Sequences. [0192] Protein sequences identified by LC-MS/MS proteomic analysis are shown in FIG. 3A through FIG.3C.
  • FIG.4A presents a multiple sequence alignment of EMSL1 (Q39487), EMSL2 (A5HMM7), EMSL3 (PDBID 5T20), EMSL5 D1 and EMSL5 D10 (R9RL27), EMSL8 (Q43418) obtained using MUSCLE.
  • FIG.4B is a ECTI1 [Q39488], and ECTI2 [Q39489] pairwise alignment, separated to illustrate the similarities between the two clusters of proteins.
  • FIG.4C shows a Neighbor Joining tree calculated from the multiple sequence alignment of EMSL1 (Q39487), EMSL2 (A5HMM70), EMSL3 (PDBID 5T20), ECEA1 and ECEA2 (R9RL27), EMSL8 (Q43418), ETCI1 (Q39488), and ETCI2 (Q39489) proteins.
  • Engineering of these sequences can focus on amino acid residues ( ⁇ 10%) that are outside of the carbohydrate binding sequences (CBSs) or other regions of the protein that are known to contribute to one or more activities.
  • the tree was calculated based on the average distance of all seven identified protein sequences using Blosum62 matrices.
  • Example 5 Active components of TE, isolated, identified and produced by Recombinant Methods.
  • the active components contained in a gel permeation chromatography purified fraction of TE were isolated and identified for first time as three taro proteins from the GNA- related superfamily were found by N-terminal sequencing: taro 12 kDa storage protein (GenBank accession: BAA03722), the tarin protein (GenBank accession: CAA53717), and the lectin protein (GenBank accession: ABQ32294).
  • a signal peptide was removed at the N-terminus by cleavage at the SAVA- LGTN sequence followed by a second cleavage step at the FR-NIP sequence, near the middle of the full-length gene product.
  • the lectin proteins all harbor a consensus carbohydrate recognition sequence (CRS) in each of the processed fragments (two CRS domains/full-length protein).
  • This sequence is QXDXNXVXY- (SEQ ID NO:2), found in Q39487, A5HMM7, R9RL27, Q43418, and B5LYJ9 var. L119F/R131P).
  • Bold letters highlight site amino acid conservation and X can be any residue.
  • Example 6 Proteomic analysis of TE by LC-MS/MS.
  • a total of 135 peptides were identified from TE trypsin/chymotrypsin cleavage using Byonic TM software and manual spectra validation. The analysis allowed unique assignment of the peptides to at least ten isolectins, complementing previous results reported for TE by N-terminus amino acid analysis.
  • TE was determined to be highly represented (>90% amino acid coverage) by lectin or more recently named mannose specific lectin MSL 2 (A5HMM7, GenBank Accession: EF541132.1) and by taro storage protein EMSL1 (aka 12kDa storage), also named mannose specific lectin 1 EMSL1 (Q39487, GenBank accession: D16173.1) from C. esculenta, with 60% amino acid coverage.
  • EMSL1 aka 12kDa storage
  • mannose specific lectin 1 EMSL1 Q39487, GenBank accession: D16173.1
  • Peptides corresponding to a composition of these two lectins previously denominated as tarin lectin (PDB ID 5T20, 5J76) was recognized with the identification of the unique peptide TIWSSNSSSK (SEQ ID NO:3; A5HM7 variant).
  • this lectin is referred to here as mannose specific lectin 3 (formed by EMSL2 (24-234)- EMSL1 (144-253)-E212K/S223N/Y224S/V240F/P25A).
  • EMSL2 24-234
  • EMSL1 144-253
  • E212K/S223N/Y224S/V240F/P25A Two variants of tuber agglutinin CEA from C. esculenta were found with >80% of amino acid coverage.
  • FIG.21 is a table showing a list of protein sequences engineered EMSL1-EMSL9 and ETCI1-ETCI3 based on proteomic analysis performed on TE samples and similarity searches using UniProt TM database (uniprot.org). From further analysis, other engineered proteins, can be added.
  • EMSL5 D10 was composed by EMSL5 D1 (24-252)-S164N/D209E/S217R.
  • these two lectins contain unique peptides that can be uniquely assigned to each one Attorney Docket No.: 15024-372PC0 Patent of them, i.e. TIWSSSSSSK (from CEA1; SEQ ID NO:13; (R9RL27) and GELIIKEDDFK (from CEA2; SEQ ID NO:50; R9RL27 – K208E Variant) peptides.
  • Globulins G2a and G2b are 89% and 96% similar to 25 kDa storage proteins Q39489 and Q39488, respectively.
  • Analyses of LC-MS/MS data identified three new peptides that exclusively belong to G1a, G1b, and G2b globulins. This finding suggested the presence of three additional isolectins in taro extract.
  • Mannose- binding lectin from Remusatia vivipara is a well characterized beta-prism II lectin (B5LYJ9, GenBank Accession: EU924066.1) and close homologue to EMSL2 (A5HMM7, GenBank Accession: EF541132.1) from C. esculenta (93% identity) and tuber agglutinin CEA (R9RL27, GenBank Accession: JX435122.1) from C. esculenta (94% identity).
  • This peptide like mannose-specific lectins from C. esculenta, undergo an identical two-step maturation process as TE proteins.
  • the peptide search analysis revealed the existence of two unique peptides in the RVL sequence (LVVLGPSVFK (SEQ ID NO:7; from B5LYJ9 var. L11F/R131P) and VLILQDDGF (SEQ ID NO:8; from B5LYJ9 var. L11F/R131P)) that were not present in C. esculenta sequences. These results suggested that a lectin containing these two peptides also could be present in TE as well. See FIG.5. All the recognized isolectins, were previously characterized to undergo an identical two-step processing as TE proteins.
  • the isolectins proteins (MSL1, EMSL2, EMSL3 , EMSL5 D1 , EMSL5 D10 , EMSL8, and EMSL9) harbor a consensus carbohydrate recognition sequence (CRS: -QXDXNXVXY, SEQ ID NO:2) in each of the processed fragments (or two CBS domains/full-length protein).
  • CRS consensus carbohydrate recognition sequence
  • Many Attorney Docket No.: 15024-372PC0 Patent of these N-glycan structures are expressed on hematopoietic and CSCs which may underlie the immune modulatory and direct anti-tumor effects, respectively.
  • MSLs Mannose Specific Lectin
  • Bacterial lysates were prepared in buffer containing 50 mM Tris-HCl (pH 8), 500 mM NaCl, 8 M Urea, 5 mM beta-mercaptoethanol (BME), 0.5 mM AEBSF, DNase solution, 10 mM MgCl 2 , 10 mM EDTA and sonicated. Cell debris were removed by centrifugation at 15,000 x g for 45 minutes at 4 °C.
  • the sample was filtered and loaded onto a previously equilibrated HiPrep 16/60 IMAC column with buffer A (15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME) and B (buffer A plus 1 M Imidazol).
  • buffer A 15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME
  • B buffer A plus 1 M Imidazol
  • Sumo-fused rMSL protein eluted fractions (molecular weight ⁇ 45kDa) were pooled and refolded on column decreasing urea concentration step-wise using buffer 15 mM Tris- HCl (pH 8), 500 mM NaCl, 5 mM BME, 1% Glycerol. Cleavage of the Sumo fusion protein was performed at 4 °C by addition of 150 ⁇ L of 1mM Ulp1 protease to refolded Sumo-MSL protein. The totality of the cleavage reaction was checked by SDS-PAGE.
  • a second HiPrep 16/60 IMAC column was performed to remove the His-tagged Sumo protein and the His-tagged Ulp1 protease.
  • TSP was collected in the flowthrough (>95% purity) as it was expected and verified by SDS-PAGE.
  • rMSL protein was concentrated and injected onto a Superdex S200-PG size exclusion column previously equilibrated with buffer 10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM TCEP. Fractions containing rMSL protein (>99% pure) were eluted from the S200 column (fractions 13-15) and identified using SDS-PAGE.
  • a size exclusion column S200 was calibrated with a gel filtration standard kit (Biorad TM ), containing thyroglobulin, bovine ⁇ -globulin, chicken ovalbumin, equine myoglobin, and vitamin B12, with molecular weights 670,000; 158,000; 44,000; 17,000, and 1,350 g/mol, respectively.
  • Biorad TM gel filtration standard kit
  • the active components had been isolated from a gel permeation chromatography purified fraction of TE.
  • FIG.2A illustrates TE chromatographic profile corresponding to the elution from Superdex S200 size exclusion column. The TE sample used was previously purified as described (by methodology in United States Patent, 8,865,642).
  • FIG.2B illustrates how TE samples (previously prepared as described in US patent 8,865,642) resolves mainly in 3 bands when it is run on an 4-20% denaturing SDS-PAGE.
  • LC-MS/MS revealed that all of the nine undergo identical processing to give two protein fragments of approximately 12-13 kDa per taro protein.
  • a signal peptide was removed at the N-terminus by cleavage at the SAVA- LGTN sequence followed by a second cleavage step at the FR-NIP sequence, near the middle of the full-length gene product.
  • the lectin proteins SEQ ID NO:12 (Q39487); SEQ ID NO:10 (A5HMM7); SEQ ID NO:13 (R9RL27); SEQ ID NO:16 (Q43418); and SEQ ID NO:17 (B5LYJ9 var.
  • L11F/R131P all harbor a consensus Carbohydrate Recognition Sequence (CRS: QXDXNXVXY; SEQ ID NO:2) in each of the processed fragments (or two CRS domains/full-length protein), where bold letters highlight site amino acid conservation and X can be any residue.
  • CRS Carbohydrate Recognition Sequence
  • lectins were highly pH stable and bind complex N-glycans (see FIG.7 and FIG.8, discussed below). Many are expressed on hematopoietic and cancer cells with stem cell properties (CSCs), which may underlie the immune modulatory and direct anti-tumor effects, respectively.
  • CSCs stem cell properties
  • Example 8 Analysis of Labeled TE Binding on the 100 Glycan Microarray and Top 15 ranked glycan structures TE N-glycan microarray analysis.
  • Fluorescently labeled TE NHS-Alexa555 was incubated on the glycan array for 1 hour at room temperature. After washing, the slide was analyzed and fluorescence was Attorney Docket No.: 15024-372PC0 Patent detected (Innopsys Innoscan TM 710 ). Data is reported in FIG.7A as the average relative fluorescence units (RFU) for each glycan structure identified, with standard deviation of the mean indicated in error bars, by a number on the x-axis.
  • REU relative fluorescence units
  • FIG.7D illustrates fluorescence of high-mannose glycans (green labels) on the printed 100-glycan microarray during the process of fluorescence detection.
  • FIG.7B shows the optimization of glycan array conditions at different TE concentrations (red labels). The data plotted in FIG.7A corresponds to 1.9 mg/mL of TE on the 100-Glycan microarray. RFU values at different TE concentrations are in red.
  • FIG.7C shows the calculation of apparent KD for Man-5 and Man-6 data. Glycans are rendered using the Symbolic Nomenclature for Glycans [SNFG].
  • AF555-TE at concentration of 0.4 ⁇ g/mL was assessed for N-glycan recognition pattern which consists of 100-N glycans in replicates of 8.
  • fluorescently labeled TE NHS-Alexa555
  • NHS-Alexa555 fluorescently labeled TE
  • the results are expressed by the average relative fluorescence units (RFU) for each glycan structure identified with standard deviation of the mean indicated in error bars and top 15 ranked glycan structures are illustrated with its respective glycan number.
  • Glycans structures are drawn using DrawGlycan-SNFG.
  • FIG.8A, FIG.8B, FIG.8C, and FIG. 8D for results.
  • Glycans are rendered using the Symbolic Nomenclature for Glycans (SNFG).
  • FIG.8B illustrates fluorescence of N-glycans on the printed 100 N-glycan microarray during the process of fluorescence detection.
  • FIG.8C shows glycan classification analyzed by MotifFinder.
  • Glycans are grouped by motif and motif families. Individual glycans are given as points on the plot. A-section corresponds to terminal mannose and terminal beta-galactose; B-section includes terminal type-2 LacNAc and sialyl terminated N- glycans; C-section corresponds to mannose N-glycans; and last, section labeled 0, are all the glycans that do not show binding.
  • FIG.5 provides a list of TE motifs. See Symbol Nomenclature for Glycans (SNFG) for complete key at ncbi.nlm.nih.gov/glycans/snfg.
  • FIG.9 relates to TE inhibits experimental and spontaneous metastasis in two TNBC models. In four independent experiments, TE profoundly prevented (98-99%) lung tumor colonization.
  • mice When tumors measured 18 mm average diameter, mice were euthanized, and extent of metastatic disease was assessed. Mice were transplanted with 500,000410.4 tumor cells in the right inguinal mammary gland on day zero. TE treatment reduced spontaneous lung metastases from mammary-gland implanted tumors by 85% (see FIG.9C) in spite of the minimal impact on the overall size of primary tumors. See FIG.9D, which shows primary tumor growth in mice from treatment in FIG.9C (P values are from a Wilcoxon exact two-sided test at 0.05 level of significance). [0227] In an independent experiment, survival time (after i.v. tumor cell injection) was extended significantly by TE.
  • TE specifically targets a process relevant to metastasis, but it does not affect primary tumor size, suggesting that it is CSC that are targeted.
  • TE also profoundly inhibited metastasis to the brain, liver and bone (not shown). Due to the pH stability of TE, it is fully active when given by oral gavage, which can facilitate clinical trials.
  • Example 10 Method of Making Recombinant EMSL1.
  • Recombinant Mannose Specific Lectin 1 (rEMSL1), previously known as Taro storage protein (rTSP) was codon optimized for expression in E. coli and subcloned into pSUMO expression vectors.
  • Bacterial lysates were prepared in buffer containing 50 mM Tris-HCl (pH 8), 500 mM NaCl, 8 M urea, 5 mM beta-mercaptoethanol (BME), 0.5 mM AEBSF, DNase solution, 10 mM MgCl2, 10 mM EDTA and sonicated. Cell debris were removed by centrifugation at 15,000 x g for 45 minutes at 4 °C. The sample was filtered and loaded onto a previously equilibrated HiPrep 16/60 IMAC column with buffer A (15 mM Tris-HCl (pH8), 500 mM NaCl, 8 M Urea, 5 mM BME) and B (buffer A plus 1 M Imidazol).
  • Sumo-MSL1 was eluted at 200 ⁇ 20 mM Imidazole and analyzed by SDS-PAGE.
  • Sumo-fused rEMSL1 eluted fractions (molecular weight ⁇ 45kDa) were pooled and refolded on column decreasing urea concentration step-wise using buffer 15 mM Tris-HCl (pH 8), 500 mM NaCl, 5 mM BME, 1% Glycerol.
  • Cleavage of the Sumo fusion protein was performed at 4 °C by addiction of 150 ⁇ L of 1mM Ulp1 protease to refolded Sumo-MSL1 protein. The totality of the cleavage reaction was checked by SDS-PAGE.
  • EMSL1 was collected in the flowthrough (>95% purity) as it was expected and verified by SDS-PAGE.
  • rEMSL1 was concentrated and injected onto a Superdex TM S200-PG size exclusion column previously equilibrated with buffer 10 mM Tris- HCl, pH 7.0, 150 mM NaCl, 1 mM TCEP. Fractions containing EMSL1 (>99% pure) were eluted from the S200 column (fractions 13-15) and identified using SDS-PAGE.
  • the yield of rEMSL1 purification was typically 7.5-10 mg of purified protein per liter of bacterial cell Attorney Docket No.: 15024-372PC0 Patent culture.
  • Size exclusion column S200 was calibrated with a gel filtration standard kit (Biorad TM ), containing thyroglobulin, bovine ⁇ - globulin, chicken ovalbumin, equine myoglobin, and vitamin B12, with M.W.670,000; 158,000; 44,000; 17,000, and 1,350 g/mol, respectively.
  • Example 11 Recombinant EMSL1 inhibits metastasis.
  • the ability of rEMSL1 to inhibit metastasis was determined in two syngeneic, highly metastatic models of Triple Negative Breast Cancer (66.1; 410.4).
  • the choice of bacteria to produce recombinant rEMSL1 was based on the fact that, by mass spectroscopy, HCD and CID MS 2 spectra of glycopeptides (evaluated for the glycan neutral loss pattern) showed that the glycosylation level of TE was very low (1% or lower).
  • FIG.10A shows the relative fluorescence units (y-axis) of TE (blue bar) and MSL1 D34 (residues 28-253; red bar) on the 100 N-glycans array (x-axis). Complex-type N-glycans were observed, but there was no evidence of O-glycosylations.
  • FIG.10 which is a comparison of 100 N-Glycan microarray analyses obtained for TE and EMSL1 28-253 .
  • FIG.10B and FIG.10C illustrate fluorescence of symmetric biantennary, asymmetric biantennary, and high-mannose N- glycans for TE (FIG.10B) and EMSL1 D34 (residues, 28-253) (FIG.10C) on the printed 100 N-glycan microarray library (ZBiotech 100N-glycan) during the process of fluorescence detection.
  • PBS, TE (200 ⁇ g/animal/day) or rEMSL1 (40 ⁇ g/animal/day) was injected intraperitoneally into syngenic Balb/cByJ female mice on days 1-4. On day 4, 3 x 10 5 line 410.4 tumor cells were injected into the lateral tail vein. Treatments continued daily for an additional 6 days. On day 17 post tumor cell injection, when control (PBS)-treated animals became moribund, mice were euthanized and surface lung tumor colonies were counted under a dissecting microscope. As reported previously and confirmed here, treatment with TE led to 99% inhibition of tumor colonization of the lungs (see FIG.6). Treatment of mice with rEMSL1 resulted in 69% inhibition of lung colonization.
  • rEMSL1 inhibited metastasis by 50%, 55%, 69% and 78% relative to control-treated mice.
  • the Y64A mutant did not compete with recombinant eGFP-EMSL1 for binding to mammary tumor cells by flow cytometry or in glycan binding assays (FIG.11).
  • Eliminating the ability of rEMSL1 to bind glycans should abolish the anti-metastatic activity.
  • rEMSL1 constructs were engineered with a mutated CBS domain; (1) rEMLS1-Y64A and (2) rEMSL1- Q56A/D58A/N60A/V62A/Y64A.
  • the latter contains five residues in the CBS domain mutated to Ala (Q ⁇ A; D ⁇ A; N ⁇ A; V ⁇ A; Y ⁇ A).
  • Balb/cByJ female mice treated daily with rEMSL1 D1 (50 ⁇ g protein) (SEQ ID:9; #001) or mutant rEMSL1 D1 Y64A protein for 10 days.
  • mice injected with 1 x 10 5 66.1-luciferase cells and bioluminescent imaging conducted on days +1, +7, +10, +14, +17 relative to tumor cell injection, p 0.018.
  • the experimental results are shown in FIG.12, which shows only the results with the Y64A mutant.
  • the data were reported as photons in lungs FIG.12A.
  • Surface lung tumor colonies at necropsy are shown in FIG.12B.
  • Example 12 Recombinant EMSL1 and TE thermal denaturation.
  • EMSL1 GeneBank accession: BAA03722
  • FIG.1 The next studies focused on rEMSL1, however, other promising TE lectins examined thus far were pursued with similar approaches.
  • Tarin lectin (EMSL3), a taro- derived lectin previously identified, as well as TE and rEMSL1, bind to high mannose N- glycans. See FIG.7 and FIG.8.
  • TE and rEMSL1 bind to Lewis x type carbohydrate structures that are frequently overexpressed on cancer cells. See FIG.10D and FIG.13C.
  • CSC cancer stem cells
  • N-glycan fucosylation of the terminal antennae i.e. Lewis X
  • resulted in increased binding affinity i.e. N004, N054, N214
  • non-fucosylated counterparts i.e. N001, N051, N211.
  • Sialylation or addition of N-acetylneuraminic acid (Neu5Ac) to terminal glycoprotein i.e. sialyl Lewis X
  • rEMSL1 bound to complex symmetrical and asymmetrical biantennary N-glycans with similar intensity, but rEMSL1 binding to high- mannose N-glycans was limited as compared to lectins reported by other groups.
  • the two CBS binding mutants rEMSL1-Y64A and rEMSL1-5xAla did not exhibit any specific binding to any glycan on the array. See FIG.13B.
  • the TE and rEMSL1 glycan microarray analysis revealed the high specificity and selectivity that TE lectins exhibit towards complex fucosylated and sialylated N-glycans, important biosignatures of TNBC and HER-2+ human breast cancers.
  • a single lectin from TE, rEMSL1 could mimic this glycan-binding profile as well as the anti-metastatic activity (see FIG.8, FIG.13, and FIG.6).
  • Example 13 Engineered rEMSL1 Characteristics.
  • the engineered rEMSL1 can be produced in large quantities with improved bioavailability and superior anti-metastatic activity when benchmarked versus wild type (wt) rEMSL1.
  • Wild type rEMSL1 has been structured by X-ray crystallography, and some dynamic features have been determined by NMR. See FIG.1H; sharp contours in 8.0 to 8.5 ( 1 H region).
  • rEMSL1 also has significant anti-metastatic activity in vivo that is not observed in constructs mutated on the glycan binding region (see FIG.12).
  • FIG.4D shows stable structural domains, but some regions of dynamic character were observed by NMR (see FIG.1H, an Attorney Docket No.: 15024-372PC0 Patent 15 N-TROSY NMR spectrum of rEMSL1 28-253 in solution showing good dispersion of NMR resonances is observed).
  • the glycan binding motifs that most readily interact with rEMSL1 have been characterized (see FIG.10 or FIG.13).
  • FIG.4D shows the X-ray structure of rEMSL1 in ribbon (green) highlighting the disulfide bond (yellow) and the carbohydrate binding sites (CRS#1 and #2) in red.
  • Glycan binding to rEMSL1 as grouped by structurally similar motifs (A, B, C, 0) is shown in FIG.8C.
  • Motif A represents N-glycans having terminal type-2 lactosamines (LacNAc), terminal N-Acetyl glucosamines (GlcNAc)
  • Motif B green represents Lewis X type N-glycans
  • Motif C cyan represents N-glycans having terminal mannose
  • motif 0 purple represents glycans that do not bind rEMSL1.
  • Example 15 100 N-Glycan Microarray Binding Assays (TE).
  • TE N-Glycan Microarray Binding Assays
  • the CBS domain is critical to the anti-cancer and glycan-binding activities, consistent with results known from the prior art.
  • FIG.10 shows 100 N-Glycan microarray binding assays for TE and rEMSL1 28-253 .
  • FIG.10A presents the relative fluorescence units (RFU) (y-axis) of TE (blue bars) and rEMSL1 (red bars) on the Attorney Docket No.: 15024-372PC0 Patent 100 N-glycans array (x-axis).
  • the data is presented as the normalized mean + s.d. of three replicates corrected by background fluorescence reading.
  • FIG.10B illustrates fluorescence of symmetric biantennary, asymmetric biantennary, and high-mannoe N-glycans binding to TE (FIG.10B) and EMSL1 D34 (residues 28-253) (FIG.10C) on the printed 100 N-glyan microarray (ZBiotech TM 100 N-glycan) during the process of fluorescene detection FIG.10C.
  • Representative N-glycan motifs were analyzed by MotifFinder TM .
  • the red shadowed fucosylated antenna also known as Lewis X carbohydrate, is a characteristic biomarker in cancer.
  • Example 16 100 N-Glycan Microarray Binding Assays (rMSL1).
  • FIG.13A presents a 100 N-Glycan microarray binding assay for rEMSL1 (28-253) (blue bar) and FIG.13B illustrates rEMSL1 mutant proteins rEMSL1-Y64A (orange bar) and rEMSL1-5xAla (green bar).
  • Bar charts show Relative Fluorescence Units (RFU) (on y-axis) for selected N-glycans by their order of glycan ID (on x-axis). The data are presented as normalized mean+standard deviation of three replicates corrected by background fluorescence reading. Each experiment was repeated three times.
  • RSU Relative Fluorescence Units
  • FIG.13C shows representative N-glycan structures showing binding to rEMSL1: Complex type biantennary N-linked glycans as N001; Lewis X type N-glycans as in N224 (circled in red); Terminal Type 2 N-acetyllactosamine (LacNAc) as in N6030 (circled in blue).
  • TE binds to high-mannose type N-glycans as Man-5. Automated analysis of glycan array data was performed using MotifFinder TM .
  • the red shadowed fucosylated antenna is a characteristic biomarker in cancer, also known as Lewis X carbohydrate.
  • FIG.14 presents competitive binding studies with line 66.1 cells and recombinant eGFP-fused-EMSL1 in the presence or absence of increasing concentrations of unlabeled rEMSL1. This experiment examines binding of recombinant eGFP-EMSL1 in presence of unlabeled rEMSL1, rEMSL1-Y64A or rEMSL1-5xAla.
  • Line 66.1 tumor cells were incubated Attorney Docket No.: 15024-372PC0 Patent with recombinant eGFP-EMSL1 or unlabled rEMSL1. After incubation, cells were washed and fluorescence intensity analyzed by FACSCanto II cytometer and data analyzed with FlowJo software. Unlabeled rEMSL1 (400, 400 ug) was able to compete with recombinant eGFP-MSL1 (40 ug) for binding to live cells. rEMSL1 D1 is able to compete with labeled rEMSL1 D21 (eGFP-EMSL1 D1 ) for binding to live murine 66.1 cells.
  • FIG.11 examines binding of recombinant eGFP-rEMSL1 in presence of unlabeled rEMSL1, rEMSL1-Y64A or rEMSL1-5xAla.
  • rEMSL1 was able to inhibit proliferation in a dose-dependent manner rMSL125 ⁇ g/ml vs PBS, p ⁇ 0.006; rEMSL1 50 ⁇ g/ml, p ⁇ 0.002; rEMSL175 ⁇ g/ml, p ⁇ 0.0004.
  • PBS, TE or rEMSL1 D1 SEQ ID NO:9; #001 was added.
  • Example 20 TE and rEMSL1 inhibit cancer cells with stem-like properties.
  • CSC breast cancer stem cell
  • the potent anti- metastatic activity of TE and rEMSL1 could be related, in part, to direct inhibition of CSC.
  • Tumorsphere TM assays were performed in serum-free MammoCult TM medium. lIne 66.1 cells plated in ultra-low attachment plate and PBS, TE or rEMSL1 D1 (SEQ ID NO:9; 1) added at time of plating. Eight days later, spheres were collected, dissociated using trypsin and cell Attorney Docket No.: 15024-372PC0 Patent number/well calculated using trypan blue.
  • PD-L1 and PD-1 represent an immune checkpoint pair that can lead to inhibition of potential anti-cancer immune responses. See FIG.20. We determined the ability of rEMSL1 (A) or TE (B) to bind to either PD-L1 or PD-1.
  • MSL1, EMSL1 and rEMSL1 D1 (UniProt:Q39487; #001) 10 MSL2, EMSL2 and rEMSL2 D1 (UniProt:A5HMM7; #201) 11 MSL3, EMSL3 and rEMSL3 D1 (PDB ID:5T20; #301) 12 MSL4, EMSL4 and rEMSL4 D1 (PDB ID:5J76; UniProt: Q39487- variant H81Y/G98K/V110D/S120D/S129G/Y210K/Y220S/V236F) 13 MSL5, EMSL5 and rEMSL5 D1 (UniProt:R9RL27; #501) Attorney Docket No.: 15024-372PC0 Patent 14 MSL6, EMSL6 and rEMSL1 D1 (UniProt:R9RL27; #501) Attorney Docket No.: 15024-372PC0 Patent 14 MSL6, EMSL6 and
  • Van Damme et al. The Major Tuber Storage Protein of Araceae Species 1s a Lectin. Plant Physiology 1995, 107, 1147-1158.
  • Kreike et al. Genetic diversity of taro, Colocasia esculenta (L.) Schott, in Southeast Asia and the Pacific. Theor Appl Genet 2004, 109 (4), 761-8.
  • Nunes et al. DNA barcoding assessment of the genetic diversity of varieties of taro, Colocasia esculenta (L.) Schott in Brazil. In Breeding and Genetic Engineering - The Biology and Biotechnology Research, Ltd, i. P., Ed. iConcept Press Ltd: 2015. 4.
  • Sindhura et al. High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells. Biomed Pharmacother 2017, 93, 654-665. 45. Shetty et al., structure of a beta-prism II lectin from Remusatia vivipara. Glycobiology 2012, 22 (1), 56-69. 46. Vajravijayan et al., Structural analysis of beta-prism lectin from Colocasia esculenta (L.) S chott. Int J Biol Macromol 2016, 91, 518-23. 47.

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Abstract

La présente invention concerne une protéine recombinante isolée dérivée d'un extrait de la corme de la plante taro, Colocasia esculenta. Il s'est avéré que l'activité antimétastatique de cette protéine est puissante contre une malignité agressive et qu'une activité spécifique est supérieure à celle de la composition de protéine de réserve de taro de l'état de la technique. L'invention concerne donc les compositions protéiques décrites, des compositions pharmaceutiques comprenant les protéines recombinantes, et des méthodes de traitement du cancer, par exemple du cancer du sein, à l'aide des protéines recombinantes ou des compositions pharmaceutiques comprenant les protéines recombinantes.
PCT/US2024/031797 2023-05-30 2024-05-30 Inhibition de métastase avec des produits végétaux recombinants Pending WO2024249713A2 (fr)

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WO2011115651A2 (fr) * 2010-03-16 2011-09-22 University Of Maryland, Baltimore Produits végétaux naturels pour le contrôle de métastase cancéreuse

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