albizzia julibrissin agglutinin isoforms

Abstract

Disclosed herein are techniques for utilizing albizia julibrissin one of four different isoforms of a seed lectin (MASL) to treat various conditions. For example, methods for reducing tumor size and reducing vascularization in a subject are provided. The methods can include administering to the subject an isolated lectin that binds sialic acid. The lectin can comprise an amino acid sequence as set forth in Isoform 1 ([SEQ ID NO: 2]), Isoform 2 ([SEQ ID NO: 3]), Isoform 3 ([SEQ ID NO: 4]), or Isoform 4 ([SEQ ID NO: 5]), thereby reducing tumor size and reducing vascularization. Administration can be via any appropriate route, such as oral or parenteral for example (intravenous, subcutaneous, or intramuscular). Also disclosed are compositions of matter, which can include the disclosed lectin in a mixture, or can be the disclosed lectin fused to at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is an affinity tag, an epitope tag, and / or an immunoglobulin.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 537,963, filed September 12, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Declaration of sequence list This application contains a sequence list electronically submitted in ST.26 XML file format, created on August 22, 2024, named SENTRI001_SL.xml, and measuring 15.329 bytes. This ST.26 XML file is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure involves the use of Mountain locust ( Maackia amurensis A combination of substances and techniques for treating various conditions such as cancer by using specific isoforms of seed lectin (MASL). Background Technology

[0004] This section aims to introduce the reader to various aspects of the technology that may relate to the aspects of the invention described below and / or claimed. This discussion is intended to help provide the reader with background information to facilitate a better understanding of the various aspects of the invention. Therefore, it should be understood that these statements should be read in this context and not as an endorsement of prior art.

[0005] Mountain locust Lectins can be used for research on binding to sialic acid residues on proteins and as a botanical agent. For example, Mountain locust Seed lectin (MASL) targets the sialic acid-modified flatfoot protein (PDPN) receptor to inhibit inflammation of arthritis chondrocytes and suppress tumor cell growth and motility. Summary of the Invention

[0006] The following addresses various deficiencies in the prior art through the disclosed material compositions and techniques.

[0007] In various aspects, a method for reducing tumor cell growth and migration can be provided. The method may include contacting tumor cells with an effective amount of a sialic acid-binding lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby reducing tumor cell growth and migration.

[0008] In various aspects, a method for reducing tumor size and angiogenesis in a subject can be provided. The method may include administering to a subject in need of this method a separated lectin bound to sialic acid, the lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby reducing tumor size and angiogenesis. The lectin may be administered parenterally. The lectin may be administered orally.

[0009] In various aspects, a method for treating cancer can be provided. This method may include administering to a subject in need an effective amount of an isolated lectin bound to sialic acid, the lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby treating the subject's cancer. The lectin may be administered parenterally. The lectin may be administered orally.

[0010] In various aspects, a method for targeting Pdpn expressed on cells can be provided. The method may include administering to a subject in need a composition comprising a sufficient amount of an isolated lectin containing the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The method may include allowing the lectin sufficient time to bind to cellular Pdpn, thereby forming a lectin-Pdpn molecular complex containing at least a portion of the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The formation of the lectin-Pdpn molecular complex inhibits the activity of Pdpn. The formation of the complex can alter cellular behavior compared to cells not exposed to the lectin. Subjects requiring this treatment may have, for example, cancers such as carcinoma, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer. The lectin can be administered parenterally. The lectin can also be administered orally.

[0011] In various aspects, a method for targeting Pdpn expressed on cells can be provided. The method may include providing a cell culture comprising tumor cells and / or normal cells. The method may include subjecting the cells to a sufficient amount of isolated lectin comprising the amino acid sequence of isoform 1 (SEQ ID NO:2) (SEQ ID NO:3) (SEQ ID NO:4) (SEQ ID NO:5) (SEQ ID NO:5) (SEQ ID NO:6) (SEQ ID NO:7) (SEQ ID NO:8) (SEQ ID NO:7 ... This method may include identifying Pdpn-overexpressing cells by immunoassay (e.g., via immunofluorescence microscopy). Tumor cells may be cancer cells, and the cancer may be, for example, carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

[0012] In various aspects, a method for inhibiting cancer cell growth can be provided. This method may include identifying cells overexpressing Pdpn and contacting the cells with an effective amount of a sialic acid-binding lectin containing the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby reducing tumor cell growth. The cells may be cancer cells, wherein the cancer may be, for example, carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer. Cancer cell growth can be inhibited by approximately 75% compared to cancer cells not contacted with the lectin.

[0013] In various respects, a pharmaceutical composition may be provided. The composition may comprise a segregated lectin bound to sialic acid, mixed with a pharmaceutically acceptable carrier, wherein the lectin comprises an amino acid sequence as shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The composition may be formulated for parenteral administration. The composition may be formulated for oral administration.

[0014] In various aspects, a kit may be provided. The kit may include an isolated lectin bound to sialic acid, wherein the lectin comprises an amino acid sequence as shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The kit may include at least one additional suitable component, which is a pharmaceutically acceptable carrier, measuring device, buffer, diluent, filter, packaging insert with instructions for use, or a combination thereof.

[0015] In various aspects, a chimeric molecule can be provided. The chimeric molecule may comprise a lectin fused to at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is an affinity tag, an epitope tag, an immunoglobulin, or a combination thereof, and wherein the lectin comprises isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The lectin can bind sialic acid, thereby reducing tumor cell growth. The polypeptide may comprise, for example, a multihistidine tag. The polypeptide may comprise, for example, an immunoglobulin or a region thereof. The chimeric molecule may also comprise a detection agent, such as a fluorescent portion.

[0016] In various aspects, a method for targeting Pdpn expressed on cells can be provided, which includes encapsulating the cells with a composition comprising chimeric molecules as disclosed herein.

[0017] In various aspects, a method for reducing the growth of Pdpn-expressing cancer cells can be provided, comprising administering an effective amount of a chimeric molecule as disclosed herein to a subject in need of this treatment. The chimeric molecule may be administered parenterally. The chimeric molecule may be administered orally. Cancers may include, for example, carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

[0018] In various aspects, a pharmaceutical composition may be provided. The composition may comprise a lectin conjugated with sialic acid in a mixture, wherein the lectin consists of an amino acid sequence as shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The mixture may comprise pharmaceutically acceptable materials, wherein the pharmaceutically acceptable materials are flavor additives, lubricants, binders, preservatives, and / or encapsulating materials. The pharmaceutical composition may comprise at least one additional pharmaceutically acceptable material, wherein the at least one additional pharmaceutically acceptable material is a diluent, artificial colorant, flavor additive, binder, stabilizer, natural or artificial sweetener, thickener, tablet disintegrant, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, hardening agent, gelatin, tragacanth gum, methylcellulose, and / or sodium carboxymethyl cellulose. The pharmaceutical composition may be in the form of a solid dosage form, and particularly in the form of powder, tablet, pill, capsule, suppository, or dispersible granules. The composition may also be in the form of a liquid dosage form, and particularly in the form of a suspension or emulsion.

[0019] In various aspects, a method for reducing cartilage degradation in a subject can be provided. This method may include administering a pharmaceutical composition to the subject comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent bound to an α-2,3-sialic acid transmembrane glycoprotein, wherein the agent is a lectin comprising the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The α-2,3-sialic acid transmembrane glycoprotein may be a flatfoot protein (PDPN), which can be expressed by chondrocytes.

[0020] In various aspects, a method for treating an inflammatory joint disease (such as arthritis, osteoarthritis, or rheumatoid arthritis) in a subject can be provided. The method may include administering a pharmaceutical composition to the subject comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent bound to an α-2,3-sialic acid transmembrane glycoprotein, wherein the agent is a lectin comprising the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The α-2,3-sialic acid transmembrane glycoprotein may be a flatfoot protein (PDPN). The total concentration of the lectin at a local level in the joint may range, for example, from about 50 nM to about 2800 nM. Administration of the composition may reduce the baseline local level of reactive oxidative stress (ROS) in the subject. The baseline local level of ROS may be reduced by at least 10% compared to the normal baseline of a healthy subject. The baseline local level of ROS in the subject may be reduced by at least two-fold. This method may include administering a second activator to the subject. The second activator may be a nonsteroidal anti-inflammatory drug (NTHE), a corticosteroid, an opioid agonist, a tumor necrosis factor (TNF) inhibitor, a disease-modifying antirheumatic drug (DMARD), or a combination thereof. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the general description of the invention given above and the detailed description of the embodiments given below, serve to explain the principles of the invention.

[0022] Figures 1A to 1B Images are SDS-PAGE results where MASL, MAA, MAL1, MAL2, and MAM (10 ug / lane) were resolved by non-reducing (1A) and reducing (1B) 12% SDS-PAGE and visualized by staining with the molecular weight markers shown.

[0023] Figure 2 This is an image of the SDS-PAGE results, showing a magnified view of the MASL bands resolved by restoring the SDS-PAGE, where the molecular weight was calculated as shown.

[0024] Figure 3 The image shows the results of SDS-PAGE, where shrPDPN (5 ug / lane) was resolved by reverting to 12% SDS-PAGE and visualized by staining with the molecular weight markers shown.

[0025] Figure 4A and 4BImages of human OSCC tissue examined by hematoxylin and eosin (H&E) staining (4A) and immunohistochemistry (4B) to detect PDPN (scale bar = 100 μm).

[0026] Figure 5 Images of Western blot results show the expression of PDPN and GAPDH in cells treated with 0 nM, 770 nM, 1440 nM, or 2880 nM MASL for 24 hours, as well as the migration of molecular weight markers as shown in the figure.

[0027] Figure 6A This is a graph illustrating cell migration after 24 hours of treatment with 0 nM, 770 nM, 1440 nM, or 2880 nM MASL, assessed by wound healing assay (scale bar = 100 μm). Data are normalized and displayed as a percentage of the untreated control (mean + SEM, n = 3), where double and quadruple asterisks indicate p < 0.01 and p < 0.0001, respectively, as indicated by the test.

[0028] Figure 6B yes Figure 6A The four MASL processing methods considered are images at times t=0 hours and t=24 hours.

[0029] Figure 7A This is a graph showing cell viability after treatment with 0 nM, 770 nM, 1440 nM, or 2880 nM MASL for 24 hours (scale bar = 50 μm), assessed by AlamarBlue assay. Data are normalized and displayed as a percentage of the untreated control (mean + SEM, n = 3). Four asterisks indicate p < 0.0001, tested as shown.

[0030] Figure 7B yes Figure 7A The four MASL processing methods considered are applied to images at time t = 24 hours.

[0031] It should be understood that the accompanying drawings are not necessarily drawn to scale and present slightly simplified representations of various features illustrating the basic principles of the invention. Specific design features such as the sequence of operations disclosed herein, including, for example, the specific dimensions, orientations, positions, and shapes of the various illustrated components, will be determined to some extent by the specific intended application and usage environment. Some features of the illustrated embodiments have been enlarged or distorted relative to other features to facilitate visual display and clear understanding. Specifically, thin features may be thickened, for example, for clarity or illustrative purposes. Detailed Implementation

[0032] The following description and accompanying drawings illustrate only the principles of the invention. It will therefore be understood that those skilled in the art will be able to design various arrangements, which, while not explicitly described or shown herein, embody the principles of the invention and are included within its scope. Furthermore, all embodiments detailed herein are primarily intended for illustrative purposes only to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the technology, and should be understood as not being limited to such specifically detailed embodiments and conditions. Additionally, as used herein, the term "or" means non-exclusive or unless otherwise specified ( For example (Otherwise, or in an alternative). Moreover, the various embodiments described herein are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0033] The numerous innovative teachings of this application will be described with particular reference to the presently preferred exemplary embodiments. However, it should be understood that such embodiments provide only a few examples of the many advantageous uses of the innovative teachings herein. Generally, the statements in this specification do not necessarily limit any of the various claimed inventions. Furthermore, some statements may apply to some inventive features but not to others. Those skilled in the art and affected by the teachings herein will recognize that the invention is also applicable to a variety of other technical fields or embodiments.

[0034] Mountain locust The nomenclature and composition of lectins have not been definitively defined to date. As disclosed herein, these lectins can be classified into two groups. MASLs are members of one group consisting of dimer-forming subunits, which are clearly mediated by cysteine ​​residues in the carboxyl region of the protein. In contrast to MASLs, members of the other group do not dimerize under non-reducing conditions.

[0035] The 287-amino acid sequence of MASL has been determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) [SEQ ID NO:1]. The disclosed MASL contains cysteine ​​at residue 272. The disclosed MASL also contains threonine, serine, and glutamic acid at residues 223, 225, and 253, respectively, instead of serine, isoleucine, and tryptophan as reported by Ochoa-Alvarez and Van Damme.

[0036] It was also disclosed that MASL consists of four subunits or isoforms (36 kD [SEQ ID NO:2], 33 kD [SEQ ID NO:3], 28 kD [SEQ ID NO:4], and 27 kD [SEQ ID NO:5]), which have the same amino acid sequence but unique glycosylation sites. All four isoforms contain glycosylation sites at residues 61 and 113 on asparagine. Asparagine is glycosylated at residue 179 on the 33 kD and 28 kD isoforms and at residue 191 on the 36 kD, 33 kD, and 28 kD isoforms. In addition to these sites, glycosylation sites were also found on asparagine 39 on the 28 kD and 33 kD isoforms and on asparagine 165 on the 36 kD and 33 kD isoforms.

[0037] It was also disclosed that MASL isoforms can be used as agents for treating diseases involving cells expressing PDPN receptors, such as human oral squamous cell carcinoma (OSCC) cells. In various aspects, MASL isoforms can be used to treat cancers such as skin cancer, leukemia, lung cancer (such as non-small cell lung cancer), colon cancer, central nervous system (CNS) cancers, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

[0038] In various treatments, lectins can be administered via any appropriate route. In some embodiments, lectins may be administered orally. In some embodiments, lectins may be administered nasally, buccally, rectally, or locally. In some embodiments, lectins may be administered parenterally (e.g., via subcutaneous, intravenous, intramuscular, or intratumoral injection).

[0039] The lectins of this disclosure can be isolated and optionally purified using conventional methods. For example, when isolated from their natural sources, the lectins can be purified to homogeneity on a suitable immobilized carbohydrate matrix and eluted with a suitable hapten. See, Goldstein and Poretz (1986), published in The lectins. Properties, functions and applications in biology and medicine (Liener et al., eds.), pp. 33-247. Academic Press, Orlando, Fla.; Rudiger (1993) published in Glycosciences: Status and perspectives (Gabius and Gabius, eds.), pp. 415-438. Chapman and Hall, Weinheim, Germany. Alternatively, lectins can be generated via recombination methods according to established procedures. See Streicher and Sharon (2003). Methods Enzymol.363:47-77. As another alternative, lectins can be generated using standard peptide synthesis techniques or chemical cleavage methods well known in the art, based on the amino acid sequences of known lectins or those disclosed herein.

[0040] As used herein, “subject” means mammal, including, for example, humans, monkeys, or cats. Subjects who benefit from therapeutic treatment with lectins include those in whom cancer has been identified or has begun to progress or metastasize. Subjects who benefit from preventative treatment with lectins include those in whom there is a predisposition to or increased risk of developing cancer (e.g., due to genetics or exposure to carcinogens), in whom cancer has not yet formed, been established, progressed, or metastasized.

[0041] The effective amount of lectin is preferably in the range of about 0.01 mg / kg body weight to about 500 mg / kg body weight, and can be readily determined by taking into account the activity of the specific lectin administered, the route of administration, the time of administration, and other factors known to those skilled in the art. Therefore, the lectins described herein can be used as drugs to treat various cancer pathologies by inhibiting tumor cell growth and metastasis.

[0042] The in vivo effects of therapeutic compositions can be evaluated in suitable animal models. For example, xenograft cancer models in which human cancer explants or passaged xenograft tissues are introduced into immunocompromised animals (such as nude mice or SCID mice) are suitable for cancer studies and have been described (Klein et al. (1997) Nature Medicine 3:402-408). For example, WO 98 / 16628 describes various xenograft models of human prostate cancer that can reproduce the development of the primary tumor, micrometastases, and the formation of osteoblastic metastases characteristic of advanced disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis.

[0043] Lectin formulations, together with conventional additives, carriers, or diluents, can be used to prepare pharmaceutical compositions, including single doses, in the form of tablets, filled capsules, or fluids such as solutions, mixtures, emulsions, elixirs, or capsules filled with such fluids, all for oral administration, as well as suppositories.

[0044] The pharmaceutical composition may be in a solid dosage form. Solid dosage forms may be powders, tablets, pills, capsules, suppositories, or dispersible granules. The pharmaceutical composition may also be in a liquid dosage form. Liquid dosage forms may be suspensions or emulsions.

[0045] Such pharmaceutical compositions and their single doses may contain conventional ingredients or components, and such dosage forms may contain any effective concentration of the active ingredient according to the intended daily dose range. A formulation containing approximately 100-300 mg of lectin per single-dose unit may represent an appropriate concentration. However, the dosage and administration regimen for treating cancer using the aforementioned methods can vary depending on the method and the target cancer, and will generally depend on many other factors known in the art.

[0046] The pharmaceutical compositions according to the invention can be administered in a variety of dosage forms. The carrier used to produce a drug containing the lectin of the invention can include both solid and liquid substances. Solid dosage forms can include powders, tablets, pills, capsules, suppositories, or dispersible granules. The solid carrier can be one or more substances that act as a diluent, flavoring agent, solvent, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material.

[0047] In powder form, the carrier is a finely pulverized solid, including lactose, hydroxypropyl methylcellulose, and PVP, mixed with an appropriate amount of finely pulverized lectin formulation.

[0048] Suitable carriers for powder and tablet forms include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, hardening agents, gelatin, tragacanth gum, methylcellulose, and sodium carboxymethylcellulose. The term "composition" is intended to... especially This includes dosage forms in which the active ingredient is encapsulated in an encapsulating material, and any dosage form may optionally be associated with a carrier. Dosage forms may include, for example, capsules or tablets.

[0049] Suppositories are produced by melting low-melting-point wax and distributing lectins within it. The molten, homogeneous mixture is then poured into a mold and allowed to cool.

[0050] Compositions suitable for vaginal application may be in the form of tablets, tampons, creams, gels, pastes, foams or sprays, and in addition to the active ingredient, contain a suitable carrier known in the art.

[0051] Compositions in liquid form include solutions, suspensions, and emulsions, such as aqueous solutions or propylene glycol solutions, and suitable colorants, flavor additives, stabilizers, or diluents. Compositions in solid form are also included, intended to be converted into a liquid form shortly before consumption. In addition to the active ingredient, these forms may also contain artificial colors, flavorings, stabilizers, buffers, natural or artificial sweeteners, dispersants, thickeners, solubilizers, etc.

[0052] For topical application to the epidermis, lectin compositions can be in the form of ointments, creams, gels, skin lotions, or transdermal creams. Ointments and creams can be formulated with aqueous or oil-based bases, with the addition of suitable thickeners and / or gelling agents. Skin lotions can be prepared with aqueous or oil-based bases and may contain one or more emulsifiers, stabilizers, dispersants, thickeners, or fragrances.

[0053] Compositions suitable for topical application in the oral cavity include lozenges and mouthwashes, wherein lozenges contain active ingredients in an inert flavoring matrix such as sucrose and gum arabic, and mouthwashes contain active ingredients in a liquid carrier.

[0054] The solution or mixture can be applied directly to the nasal cavity using conventional methods such as drops or sprays. The composition can be produced in single-dose or multi-dose forms. Multi-dose forms will include droppers, pipettes, or nebulizers for delivering a predetermined volume of the composition.

[0055] Inhalation can be achieved through the use of aerosol formulations, in which the active ingredient is placed in a pressurized container along with a suitable delivery agent such as CFCs, trichlorofluoromethane, dichlorofluoromethane, carbon dioxide, or other suitable gases. The dosage can be controlled by an appropriate valve system.

[0056] The pharmaceutical composition is preferably provided as a single-dose unit containing an appropriate amount of the active ingredient. The single dose may be provided in a package or as a kit including a measuring device (e.g., a device for measuring oral or injectable doses, i.e., a measuring cup, needle, or syringe)). The kit may also include other materials such as buffer solutions, diluents, filters, and packaging inserts with instructions for use. A label may be present on the kit to indicate that the composition is intended for a specific therapy and may also indicate instructions for use, such as those described above.

[0057] The compositions and methods of the present invention can also be used to reduce the side effects of conventional chemoradiotherapy, wherein lectins are administered in combination with chemoradiotherapy to reduce the amount of toxic dose required to kill cells.

[0058] In various aspects, a method for reducing tumor cell growth and migration can be provided. The method may include contacting tumor cells with an effective amount of a lectin bound to sialic acid, thereby reducing tumor cell growth and migration. The lectin may comprise the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

[0059] In various aspects, a method for reducing tumor size and angiogenesis in a subject can be provided. The method may include administering a separated lectin bound to sialic acid to a subject in need, thereby reducing tumor size and angiogenesis. The lectin may comprise the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

[0060] In various aspects, a method for treating cancer can be provided. This method may include administering an effective amount of an isolated lectin bound to sialic acid to a subject in need, thereby treating the subject's cancer. The lectin may contain the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

[0061] In various aspects, a method for targeting Pdpn expressed on cells can be provided. This method may include administering the drug to a subject in need of it. For example The method may involve administering a composition containing a sufficient amount of isolated lectin to a patient suffering from cancer (such as carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer), the isolated lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The method may include allowing the isolated lectin sufficient time to bind to cellular Pdpn, thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The formation of the complex may inhibit the activity of Pdpn. In some embodiments, the formation of the complex alters cell behavior compared to cells not exposed to the lectin.

[0062] In various aspects, a method for targeting Pdpn expressed on cells can be provided. The method may include providing a cell culture comprising cells from cancers such as carcinoma, leukemia, lung cancer, colon cancer, CNS cancers, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer, and / or normal cells. The method may include subjecting the cells to a sufficient amount of isolated lectin comprising an amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The method may include allowing the isolated lectin sufficient time to bind to cellular Pdpn, thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The formation of the complex can inhibit the activity of Pdpn. This method may include identifying cells overexpressing Pdpn by immunoassay (e.g., via immunofluorescence microscopy).

[0063] In various aspects, a method for inhibiting cell growth from cancers such as carcinoma, leukemia, lung cancer, colon cancer, CNS cancers, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer can be provided. The method may include identifying cells overexpressing Pdpn and contacting the cells with an effective amount of a lectin binding to sialic acid, thereby reducing tumor cell growth. The lectin may comprise the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). In some embodiments, cancer cell growth can be inhibited by approximately 75% compared to cancer cells not contacted with the lectin.

[0064] In various aspects, a pharmaceutical composition may be provided. The pharmaceutical composition may comprise a separated lectin bound to sialic acid, mixed with a pharmaceutically acceptable carrier. The lectin may comprise the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

[0065] In some embodiments, a kit may be provided. The kit may include an isolated lectin bound to sialic acid. The lectin may contain the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The kit may include at least one additional suitable component. This additional suitable component may be a pharmaceutically acceptable carrier, measuring device, buffer, diluent, filter, packaging insert with instructions for use, or a combination thereof.

[0066] In several respects, a chimeric molecule can be provided. The chimeric molecule may comprise a lectin fused to at least one heterologous polypeptide. The at least one heterologous polypeptide may be an affinity tag, an epitope tag, an immunoglobulin, or a combination thereof. The lectin may comprise isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The lectin may bind sialic acid, thereby reducing tumor cell growth. The polypeptide may comprise a multihistidine tag. The polypeptide may comprise an immunoglobulin or a region thereof. The chimeric molecule may comprise a detection agent (such as a fluorescent portion).

[0067] In various aspects, a method for targeting Pdpn expressed on cells can be provided. This method may include contacting cells with a composition comprising an embodiment of the disclosed chimeric molecule.

[0068] In various aspects, a method for reducing the growth of cancer cells expressing Pdpn can be provided. This method may include embodiments of administering an effective amount of the disclosed chimeric molecule to a subject in need. In some embodiments, the cancer may be carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

[0069] In various aspects, a pharmaceutical composition may be provided. The pharmaceutical composition may be a mixture comprising isolated lectins bound to sialic acid. The lectin may comprise the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The mixture may comprise pharmaceutically acceptable materials. Pharmaceutically acceptable materials can be flavoring agents (e.g., oils such as peppermint oil, natural sweeteners such as sugar, or artificial sweeteners such as saccharin), lubricants (for preventing adhesion to surfaces, such as magnesium stearate, sodium stearoyl fumarate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated vegetable oils, etc.), binders (for holding products together or maintaining a specific shape, such as sugar, gelatin, gum, microcrystalline cellulose and other modified cellulose, waxes, or synthetic polymers such as polyethylene glycol, etc.), preservatives (such as benzyl alcohol, sorbic acid, methylparaben, or propylparaben, etc.), and / or encapsulating materials (e.g., poly(vinyl alcohol), poly(acrylic acid), poly(acrylamide), poly(ethylene oxide), poly(lactic acid), poly(glycolic acid), polycaprolactone, poly(lactic-co-glycolic acid), chitosan, cellulose, etc.). Such materials are well understood in the art.

[0070] The pharmaceutical composition may contain at least one other pharmaceutically acceptable material. At least one other pharmaceutically acceptable material may be a diluent, artificial colorant, flavoring additive, binder, stabilizer, natural or artificial sweetener, thickener, tablet disintegrant, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, hardening agent, gelatin, tragacanth gum, methylcellulose, and / or sodium carboxymethyl cellulose.

[0071] The pharmaceutical composition may be in a solid dosage form. Solid dosage forms may be powders, tablets, pills, capsules, suppositories, or dispersible granules. The pharmaceutical composition may also be in a liquid dosage form. Liquid dosage forms may be suspensions or emulsions.

[0072] In various aspects, a method for reducing cartilage degradation in a subject can be provided. This method may include administering a pharmaceutical composition to the subject. The pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent bound to an α-2,3-sialic acid transmembrane glycoprotein. The agent may be a lectin comprising the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The α-2,3-sialic acid transmembrane glycoprotein may be flatfoot protein (PDPN). PDPN may be expressed by chondrocytes.

[0073] In various aspects, it can provide a treatment for inflammatory joint diseases in the subject ( For example Methods for treating arthritis, such as osteoarthritis or rheumatoid arthritis. The method may include administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent bound to an α-2,3-sialic acid transmembrane glycoprotein. The agent may be a lectin comprising the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID No:5]). The α-2,3-sialic acid transmembrane glycoprotein may be flatfoot protein (PDPN). The total concentration of the lectin at a local level in the joint may be in the range of about 50 nM to about 2800 nM. In some embodiments, administration reduces the baseline local level of reactive oxidative substances (ROS) in the subject, such as by at least 10% compared to the normal baseline of a healthy subject. In some embodiments, administration reduces the baseline local level of reactive oxidative substances (ROS) in the subject, such as by at least two-fold. The method may include administering a second activator to the subject, wherein the second activator is a nonsteroidal anti-inflammatory drug (NTHE), a corticosteroid, an opioid agonist, a tumor necrosis factor (TNF) inhibitor, a disease-modifying antirheumatic drug (DMARD), or a combination thereof.

[0074] Mountain locust (Maackia amurensis) Lectins have been used in research for over 60 years. In particular, Mountain locust Seed lectin (MASL) has been used as an agent that binds to α-2-3-linked sialic acid. These glycosylation modifications are commonly found on extracellular protein receptors associated with viral infection, inflammation, and cancer [5, 6]. Therefore, MASL can target these receptors to inhibit viral infection, arthritis development, and cancer progression. For example, MASL inhibits the binding of the SARS-CoV-2 spike protein to the ACE2 receptor in cell cultures. MASL also targets the PDPN receptor to inhibit the progression of osteoarthritis and rheumatoid arthritis in mice and to inhibit excessive proliferation of psoriatic epidermis in skin cultures. In addition, MASL targets the PDPN receptor to inhibit the motility and viability of melanoma cells in cultures and tumor progression in mice. MASL also targets the PDPN receptor to inhibit the growth and motility of oral squamous cell carcinoma (OSCC) cells and is being evaluated as a treatment for oral cancer in an ongoing Phase 1 human clinical trial (NCT04188665). Mountain locust Lectins can also target receptors to inhibit the viability of non-small cell lung cancer and childhood acute lymphoblastic leukemia cells.

[0075] The supplier provides under several different names Mountain locust lectins, and their nomenclature is not clearly defined. These can be divided into two main categories: (1) MAL ( Mountain locust lectins), which include MAM ( Mountain locust mitogens), MAL1, MAA ( Mountain locust (1) lectin) and MAA1, and (2) MAH ( Mountain locust Hemagglutinins (HAGs) include MAL2, MAA, and MAA2. It is worth noting that MAA is included in both groups, and some names include MAL and MAL1, MAA and MAA1, MAH and MAL2, and MAA and MAA2 are sometimes used interchangeably. Additionally, some suppliers indiscriminately offer MAA or MAL as potential mixed lectins.

[0076] Using SDS-PAGE to parse MASL and others Mountain locust Lectins, including MAA, MAL1, MAL2, and MAM, were analyzed on 12% SDS-PAGE gels (20 μg / lane) containing (reducing) or non-reducing 10% β-mercaptoethanol loading buffer (2% SDS, 10% glycerol, and 0.05% bromophenol blue, dissolved in 62.5 mM Tris-HCl pH 6.8) and stained with COOMASSIE® Brilliant Blue R-250 (CI42660), available via, for example, Sigma-Aldrich.

[0077] Reducing gel analysis resolved MASL, MAL1, and MAM into two major bands at 36 kD and 33 kD, and two minor bands at 28 kD and 27 kD. In contrast to these three lectins, reducing gel analysis resolved MAA and MAL2 into smaller subunits, exhibiting two major bands at 28 kD and 27 kD, as shown below. Figures 1A to 1B As shown.

[0078] The results of restoring SDS-PAGE (see) Figure 1B This indicates that all the checks conducted here... Mountain locust All lectins contained subunits migrating at 28 kD and 27 kD. Then, non-reducing SDS-PAGE was performed (see...). Figure 1AThe ability of these subunits to dimerize was examined. The MASL subunit formed a dimer migrating at 72 kD, which is likely mediated by cysteine ​​residues in the carboxyl region of the protein, as previously reported. The MAL1 and MAM subunits also produced these dimers on non-reducing gels. However, the MAA and MAL2 subunits did not form dimers on these gels. Therefore, these lectins represent two distinct groups, in which MASL, MAL1, and MAM form dimers, while the group containing MAA and MAL2 does not form dimers on non-reducing gels, as shown in the figure. Figure 1A As shown.

[0079] MASL was sequenced to verify its identity. Using the reductive SDS-PAGE disclosed in this paper, the subunits were resolved at 27 kD, 28 kD, 33 kD, and 36 kD, and are shown below. Figure 1A , 1B In step 2, each strip was cut off and inspected via LC-MS / MS.

[0080] LC-MS / MS For example, the analysis was performed as described in Retzbach et al., “Independent effects of Src kinase and podoplaninon anchorage independent cell growth and migration” (2022), Craig et al., “TANDEM: matching proteins with tandem mass spectra” (2004), and Gupta et al., “Target-decoy approach and false discovery rate: when things may go wrong” (2011), with the following modifications. 10 μg MASL was analyzed by SDS-PAGE, stained with COOMASSIE® Brilliant Blue R-250, and bands were excised from the gel. Proteins were reduced with 10 mM DTT at 60 °C for 30 min, alkylated with 20 mM iodoacetamide at room temperature in the dark for 45 min, and digested overnight with 0.2 µg trypsin (37 °C), chymotrypsin (20 °C), or Asp-N (37 °C) (Pierce MS grade, ThermoFisher). Peptides were extracted twice with 5% formic acid, once with 60% acetonitrile, and dried under vacuum. Half of the chymotrypsin peptide was further digested with PNGase F. LC-MS / MS was performed using a nano LC (DIONEX™ ULTIMATE™ 3000RLSCnano system, ThermoFisher) connected to an OrbitrapEclipse Tribrid mass spectrometer (ThermoFisher). Each sample (approximately 25% of the digest) was loaded onto a fused silica trap column (ACCLAIM™ PEPMAP™ 100 HPLC column, 75 μm x 2 cm, ThermoFisher). After washing with 0.1% TFA at 5 µl / min for 5 min, the trap column was tandem with the analytical column (Nanoease M / Z peptide BEH C18, 130A, 1.7 μm, 75 μm x 250 mm, Waters) for LC-MS / MS. The peptides were fractionated using a segmented linear gradient at 300 nL / min, consisting of 4%–15% B (where A: 0.2% formic acid and B: 0.16% formic acid and 80% acetonitrile) over 5 minutes, 15%–50% B over 50 minutes, and 50%–90% B over 15 minutes. Then, before the next run, the solution B was restored to 4% for 5 minutes.The scan sequence began with the MS1 spectrum (Orbitrap analysis, resolution 120,000, scan range 275–1500 m / z, auto-gain control target 1E6, maximum injection time 100 ms). The first N (3-second) duty cycle schemes were used to determine the number of MS / MS scans to be performed for each cycle. Precursor ions with charges 1–7 were selected for MS / MS, with a 60-second dynamic exclusion period to avoid duplicate sampling. Precursor ions were separated in a quadrupole with a separation window of 1.2 m / z, auto-gain control target 1E5, and fragmented by high-energy collisional dissociation at 30% of the normalized collision energy. The fragments were scanned in Orbitrap at a resolution of 15,000 m / z. The MS / MS scan range was determined by the charge state of the parent ion, but the lower limit was set at 110 m / z. The peak list MASCOT Universal Format (MGF) file was generated by Thermo Proteome Discoverer (version 2.4) and searched using the built-in GPM Fury (X!Tandem Alanine) database, which includes relevant MASL and variant sequences, as well as the FASTA database consisting of Common Laboratory Contaminants (CRAPs). Mudpit searches were performed using all MGF files from LC-MS / MS analyses of each sample digested with different proteases. The initial search parameters were: parental quality error ± 7 ppm, fragment quality error ± 20 ppm, fixed modification of cysteine ​​urea methylation, and variable modifications of methionine monooxidation and asparagine deamination. Variable modifications during three rounds of purification were as follows: Round 1 – monooxidation at methionine and tryptophan, Round 2 – deamination at glutamine and asparagine, Round 3 – dioxidation at methionine and tryptophan, with protease specificity set to non-specificity. The minimum 5 acceptable peptide and protein expected fractions were set to 10⁻² and 10⁻⁴, respectively. Results were confirmed and analyzed by manual inspection. No evidence of O-linked glycosylation sites was found, and N-linked sites were confirmed by PNGase F digestion.

[0081] All four MASL isoforms contain the same primary amino acid sequence. This obtained sequence was compared with previously reported sequences. The sequence contains a cysteine ​​residue at residue 272 similar to those reported by Ochoa-Alvarez et al. and Yamamoto et al., but opposite to the serine residue at that position reported by Van Damme et al. This cysteine ​​residue is similar to... Figure 1AThe driving dimer formation observed in the non-reducing gel shown is consistent. The MASL sequence defined here also contains threonine, serine, and glutamic acid at residues 223, 225, and 253, instead of serine, isoleucine, and tryptophan reported by Ochoa-Alvarez et al. and Van Damme et al. Glycosylation sites were also found on all four MASL isoforms as disclosed herein.

[0082] The PDPN receptor has been identified as a biologically relevant MASL target. It is hypothesized that sialic acid on PDPN is involved in MASL binding. Soluble recombinant human PDPN (srhPDPN) was generated in human HEK293F cells to verify the sialic acid modification of this receptor.

[0083] Specifically, the sequence encoding a protein consisting of a 19-amino acid signal peptide (SEQ ID NO:7) from the V region of the mouse Ig heavy chain BCL1, followed by the entire extracellular region of human PDPN containing amino acids 23-131 (SEQ ID NO:6) terminated by an HIS tag (SEQ ID NO:8), was engineered into pcDNA3.1(+) and transfected into FREESTYLE™ HEK293F cells (ThermoFisher #R790-07). After tangential percolation of the culture medium, the resulting soluble recombinant human PDPN (srhPDPN) was purified on a Ni agarose gel, then exchanged for PBS on a SUPERDEX™ 200 increase column, and analyzed by SDS-PAGE. For mass spectrometry analysis, srhPDPN (0.5 µg / µl, dissolved in 25 mM ammonium bicarbonate, pH 8) was digested with trypsin (0.01 µg / µl) or GluC (0.01 µg / µl) (sequencing grade; Promega) at 37°C for 16 h. The digestion reaction was terminated by adding trifluoroacetic acid to 1%. The peptide mixture (10 µl) was injected and analyzed by LC-MS / MS on a DIONEX™ ULTIMATE™ 3000 RSLCnano system connected in-line to a Q EXTRACTIVE™ HFBiopharma mass spectrometer (Thermo). The sample was collected for 2 min at a rate of 20 μl / min in loading solvent A (0.1% aqueous trifluoroacetic acid) on a 5 mm trapping column (Thermo Scientific, 300 μm inner diameter (ID), 5 μm beads). Peptides were separated on a 250 mm Aurora Ultimate, 1.7 µm C18, 75 µm inner diameter (IonOpticks) spectrometer maintained at a constant temperature of 45 °C. The peptides were eluted via a nonlinear gradient, starting with 0.5% Solvent B, reaching 26% Solvent B (0.1% trifluoroacetic acid in acetonitrile) within 30 min, 44% Solvent B within 38 min, followed by washing with 56% Solvent B for 7 min, and reequilibration with Solvent A at a flow rate of 300 nl / min. The mass spectrometer was operated in data correlation mode, automatically switching between MS and MS / MS acquisition to acquire the 12 most abundant ion peaks for each MS spectrum. After accumulating to a target value of 3,000,000, full-scan MS spectra (375–1500 m / z) were acquired at a resolution of 60,000 in an Orbitrap analyzer. After filling the trap with a target value of 100,000 for a maximum of 120 ms, the 12 strongest ions above the threshold of 15,000 are separated with a width of 1.5 m / z and fragmented with 28% of the normalized collision energy.MS / MS spectra (200–2000 m / z) were obtained at a resolution of 15,000 in an Orbitrap analyzer. During the project, a polydimethylcyclosiloxane background ion of 445.120028 Da was used for internal calibration (locking in quality), and QCloud was used to control longitudinal instrument performance. Data analysis was performed using the peptide mapping tool with BioPharma Finder software (Thermo Fisher Scientific). The obtained MS / MS spectra were analyzed using BioPharma Finder 3.0 software (Thermo Fisher Scientific), and the selected trypsin or Glu-C was mapped to the appropriate protein sequence. For peptide identification, the following parameters were used: a maximum peptide quality of 7000 Da, a quality accuracy of 5 ppm, and a minimum confidence level of 0.80. Deamidation of asparagine and glutamine, and oxidation of methionine and tryptophan were set as variable modifications. Searching for glycosylation modifications (human-specific) was implemented, and the maximum number of variable modifications per peptide was set to 2.

[0084] like Figure 3 As shown, the protein migrated by SDS-PAGE with an estimated molecular weight of 24.6 kD instead of its expected size of 12 kD.

[0085] Sequencing of the protein by LC-MS / MS revealed that PDPN was glycosylated at 26 amino acids. These glycosylation events occurred only at serine or threonine residues. Seventeen of these 26 modifications contained sialic acid, including threonine glycosylation at residues 34 and 52, as previously reported.

[0086] MASL can target PDPN to inhibit the growth and motility of human OSCC cells. Figure 4A , 4B As shown in Figures 5 and 6, robust PDPN expression was found in human OSCC cells by IHC from patient biopsies and Western blots of culture-suitable cells.

[0087] For cell migration and viability assays, Sen1bs human OSCC cells were obtained from oral cancer patients participating in a clinical trial (#NCT04188665). They were maintained at 37°C in 5% CO2 and 100% humidity in DMEM (Hyclone SH30021) supplemented with 25 mM HEPES (HycloneSH30237) and FBS (Seradigm 1400–500) and grown to confluence on 6-well tissue culture cluster plates (Falcon 353224) as described [13, 14]. For migration assays, cell monolayers were scraped and observed immediately before and 24 hours after treatment with 0 nM, 770 nM, 1440 nM, or 2280 nM MASL, and migration was quantified based on the number of cells entering a 123 × 123 μm square placed along the center of the wound. Sister plates treated with MASL for 24 hours were incubated with AlamarBlue (BioRad #BUF012A) for 4 hours and measured (excitation / emission: 570 / 600 nm) to assess cell viability.

[0088] Western blotting was performed as described in, for example, Hamilton et al., “Effects of Maackia amurensis seedlectin (MASL) on oral squamous cell carcinoma (OSCC) gene expression and transcriptional signaling pathways” (2021) and Sheehan et al., “Heterocellular N-cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells” (2022). The confluent Sen1bs cells were treated with 0 nM, 770 nM, 1440 nM, or 2280 nM MASL for 24 hours, washed with PBS, transferred to microcentrifuge tubes, precipitated, aspirated, and... The protein was frozen at 80°C and then lysed in buffer (2% SDS, 10% glycerol, 10 mM EDTA, 50 nM DTT, 50 mM NaF, 0.2 mM Na3VO4 and 1 mM PMSF dissolved in 62.5 mM Tris pH 6.8), sonicated and clarified by centrifugation. Proteins were analyzed by SDS-PAGE (10 μg / lane), transferred to an Immobilon-P membrane (Millipore #1PVH00010), and incubated with antiserum specific to PDPN (D2-40 Agilent M361901-2) and GAPDH (Santa Cruz #FL335). Primary antibodies were recognized by appropriate secondary anti-IgG antibodies conjugated to horseradish peroxidase, including those from mice (Jackson Immuno Research #115-035-003) and rabbits (Proteintech #SA00001-2), and detected using enhanced chemiluminescence (Thermo Scientific 32209). The gels were stained with COOMASSIE® Brilliant Blue R-250, and the membranes were stained with Indian ink to verify equivalence of loading and transfer after blotting.

[0089] MASL inhibited cell motility and viability in a dose-dependent manner. Compared with the untreated control, MASL reduced cell motility by 61% ± 8.2%, 91% ± 2.8%, and completely decreased it at 770 nM, 1540 nM, and 3080 nM, respectively (mean ± SEM, n = 3). Figures 6A to 6B As shown. Compared with the untreated control, MASL at 770 nM, 1540 nM, and 3080 nM also reduced cell viability by 29% ± 1.5%, 48% ± 0.3%, and 59% ± 2.2%, respectively. Figures 7A to 7B As shown. However, contrary to results from OSCC cells obtained from different patients, MASL exposure did not reduce PDPN expression in these OSCC cells, as... Figure 5 As shown.

Claims

1. A method for reducing tumor cell growth and migration, comprising contacting tumor cells with an effective amount of a lectin bound to sialic acid, said lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby reducing tumor cell growth and migration.

2. A method for reducing tumor size and angiogenesis in a subject, comprising administering to a subject in need of such action an isolated lectin bound to sialic acid, said isolated lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby reducing tumor size and angiogenesis.

3. The method of claim 2, wherein the separated lectin is administered parenterally.

4. The method of claim 2, wherein the separated lectin is administered orally.

5. A method for treating cancer, comprising administering to a subject in need an effective amount of an isolated lectin conjugated with sialic acid, said isolated lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), thereby treating said cancer.

6. The method of claim 5, wherein the separated lectin is administered parenterally.

7. The method of claim 5, wherein the separated lectin is administered orally.

8. A method for targeting Pdpn expressed on cells, comprising: Administer to a subject in need a composition comprising a sufficient amount of isolated lectin, said isolated lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]); and The separated lectin is allowed sufficient time to bind to cellular Pdpn, thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]); The formation of the lectin-Pdpn molecular complex inhibits the activity of Pdpn.

9. The method of claim 8, wherein the formation of the lectin-Pdpn molecular complex alters the behavior of the cells compared to cells not exposed to the isolated lectin.

10. The method of claim 8, wherein the subject in need of this method has cancer.

11. The method of claim 10, wherein the cancer is cancer, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

12. The method of claim 8, wherein the isolated lectin is administered parenterally.

13. The method of claim 8, wherein the separated lectin is administered orally.

14. A method for targeting Pdpn expressed on cells, comprising: Provide cell cultures containing tumor cells and / or normal cells; The tumor cells are subjected to a sufficient amount of isolated lectin comprising the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]); and The separated lectin is allowed sufficient time to bind to cellular Pdpn, thereby forming a lectin-Pdpn molecular complex containing at least a portion of the amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]). The formation of the lectin-Pdpn molecular complex inhibits the activity of Pdpn.

15. The method of claim 14, further comprising identifying cells overexpressing Pdpn by immunoassay.

16. The method of claim 15, wherein the immunoassay utilizes immunofluorescence microscopy.

17. The method according to claim 14, wherein the tumor cells are cancer cells, leukemia cells, lung cancer cells, colon cancer cells, central nervous system (CNS) cancer cells, melanoma cells, ovarian cancer cells, kidney cancer cells, prostate cancer cells and / or breast cancer cells.

18. A method for inhibiting the growth of cancer cells, comprising: Identify cells overexpressing Pdpn; as well as By contacting Pdpn-overexpressing cells with an effective amount of sialic acid-binding lectin, the lectin comprising the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]), tumor cell growth is reduced.

19. The method of claim 18, wherein the cancer is skin cancer, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

20. The method of claim 18, wherein the cell growth of the cancer is inhibited by about 75% compared to cancer cells that have not been in contact with the lectin.

21. A pharmaceutical composition comprising an isolated lectin bound to sialic acid, mixed with a pharmaceutically acceptable carrier, wherein the isolated lectin comprises the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for parenteral administration.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for oral administration.

24. A reagent kit comprising: A lectin conjugated with sialic acid, wherein the conjugated lectin comprises the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]); and At least one other suitable component, said at least one other suitable component being a pharmaceutically acceptable carrier, measuring device, buffer, diluent, filter, packaging insert with instructions for use, or a combination thereof.

25. A chimeric molecule comprising a lectin fused to at least one heteropeptide, wherein the at least one heteropeptide is an affinity tag, an epitope tag, an immunoglobulin, or a combination thereof, and wherein the lectin comprises isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

26. The chimeric molecule of claim 25, wherein the lectin binds to sialic acid, thereby reducing tumor cell growth.

27. The chimeric molecule of claim 25, wherein the at least one heteropeptide comprises a multihistidine tag.

28. The chimeric molecule of claim 25, wherein the at least one heterologous polypeptide comprises an immunoglobulin or a region thereof.

29. The chimeric molecule of claim 25, further comprising a detection agent.

30. The chimeric molecule according to claim 29, wherein the detection agent is a fluorescent portion.

31. A method for targeting Pdpn expressed on cells, comprising contacting the cells with a composition comprising the chimeric molecule according to claim 25.

32. A method for reducing the growth of cancer cells expressing Pdpn, comprising administering an effective amount of the chimeric molecule according to claim 25 to a subject in need of such treatment.

33. The method of claim 32, wherein the chimeric molecule is administered parenterally.

34. The method of claim 32, wherein the chimeric molecule is administered orally.

35. The method of claim 32, wherein the cancer is cancer, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and / or breast cancer.

36. A pharmaceutical composition comprising a lectin conjugated with sialic acid in a mixture, wherein the lectin comprises the amino acid sequence shown in isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

37. The pharmaceutical composition of claim 36, wherein the mixture comprises pharmaceutically acceptable materials, wherein the pharmaceutically acceptable materials are flavor additives, lubricants, binders, preservatives and / or encapsulating materials.

38. The pharmaceutical composition according to claim 36, wherein it is in the form of a solid dosage form, said solid dosage form being a powder, tablet, pill, capsule, suppository, or dispersible granule.

39. The pharmaceutical composition of claim 38, further comprising at least one additional pharmaceutically acceptable material, wherein the at least one additional pharmaceutically acceptable material is a diluent, artificial colorant, flavoring additive, binder, stabilizer, natural or artificial sweetener, thickener, tablet disintegrant, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, hardening agent, gelatin, tragacanth gum, methylcellulose and / or sodium carboxymethylcellulose.

40. The pharmaceutical composition according to claim 36, wherein it is in the form of a liquid dosage form, said liquid dosage form being a suspension or an emulsion.

41. A method for reducing cartilage degradation in a subject, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to α-2,3-sialic acid transmembrane glycoprotein, wherein the agent is a lectin comprising an amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

42. The method according to claim 41, wherein the α-2,3-sialic acid transmembrane glycoprotein is a flatfoot protein (PDPN).

43. The method of claim 42, wherein the PDPN is expressed by chondrocytes.

44. A method of treating an inflammatory disease of a joint in a subject, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to α-2,3-sialic acid transmembrane glycoprotein, wherein the agent is an isolated lectin comprising an amino acid sequence of isoform 1 ([SEQ ID NO:2]), isoform 2 ([SEQ ID NO:3]), isoform 3 ([SEQ ID NO:4]), or isoform 4 ([SEQ ID NO:5]).

45. The method of claim 44, wherein the inflammatory disease is arthritis.

46. ​​The method of claim 45, wherein the arthritis is osteoarthritis or rheumatoid arthritis.

47. The method of claim 44, wherein the α-2,3-sialic acid transmembrane glycoprotein is a flatfoot protein (PDPN).

48. The method of claim 44, wherein the total concentration of the isolated lectin at the local level of the joint ranges from about 50 nM to about 2800 nM.

49. The method of claim 44, wherein the administration of the pharmaceutical composition reduces the baseline local level of reactive oxidative stress (ROS) in the subject.

50. The method of claim 49, wherein the baseline local level of the ROS is reduced by at least 10% compared to the normal baseline of a healthy subject.

51. The method of claim 49, wherein the baseline local level of the ROS in the subject is reduced by at least two-fold.

52. The method of claim 44, further comprising administering a second active agent to the subject, wherein the second active agent is a nonsteroidal anti-inflammatory drug (NTHE), a corticosteroid, an opioid agonist, a tumor necrosis factor (TNF) inhibitor, a disease-modifying antirheumatic drug (DMARD), or a combination thereof.

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