WO2025059258A1 - Maackia amurensis seed lectin isoforms - Google Patents

Abstract

Disclosed are techniques for treating various conditions, utilizing one of four different isoforms of Maackia amurensis seed lectin (MASL). For example, methods for reducing tumor size and vascularization in a subject are provided. The method may include administering to a subject an isolated lectin that binds sialic acid. The lectin may include 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 decreasing tumor size and vascularization. The administration may occur via any appropriate route, such as orally, or parenterally (e.g, intravenous, subcutaneous, or intramuscular). Also disclosed are compositions of matter, that may include the disclosed lectins in a mixture, or may be a disclosed lectin fused to at least one heterologous polypeptide, where the at least one heterologous polypeptide is an affinity tag, an epitope tag, and/or an immunoglobulin.

Description

MAACKIA AMURENSIS SEED LECTIN ISOFORMS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application No. 63/537,963, filed September 12, 2023, the contents of which are incorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ST.26 XML file format, created on August 22, 2024, is named SENTRI001 SL.xml and is 15.329 bytes in size. The ST.26 XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions of matter and techniques for treating various conditions, such as cancers, utilizing specific isoforms of Maackict amurensis seed lectin (MASL).

BACKGROUND

This section is intended to introduce the reader to various aspects of art, which may be related to various aspects of the present invention that are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of the various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, Maackia amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility.

BRIEF SUMMARY

Various deficiencies in the prior art are addressed below by the disclosed compositions of matter and techniques. In various aspects, a method for reducing tumor cell growth and migration may be provided. The method may include contacting a tumor cell with an effective amount of a lectin that binds sialic acid comprising 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 cell growth and migration.

In various aspects, a method for reducing tumor size and vascularization in a subject may be provided. The method may include administering to a subject in need thereof an isolated lectin that binds sialic acid, the lectin comprising 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 decreasing tumor size and vascularization. The lectin may be administered parenterally. The lectin may be administered orally.

In various aspects, a method for treating cancer may be provided. The method may include administering to a subject in need thereof an effective amount of an isolated lectin that binds sialic acid, the lectin comprising 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:5J), thereby treating the subject's cancer. The lectin may be administered parenterally. The lectin may be administered orally.

In various aspects, a method of targeting Pdpn expressed on a cell may be provided. The method may include administering to a subject in need thereof a composition comprising sufficient amounts of an isolated lectin comprising the amino acid sequence 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]). The method may include allowing sufficient amount of time for the lectin to bind cellular Pdpn thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence 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]). Formation of the lectin-Pdpn molecular complex inhibits the activity of Pdpn. Formation of the complex may modify the cells behavior as compared to cells not exposed to the lectin. The subject in need thereof may be, e.g.. suffering from cancer, such as carcinoma, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer. The lectin may be administered parenterally. The lectin may be administered orally.

In various aspects, a method of targeting Pdpn expressed on a cell may 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 an 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]). The method may include allowing sufficient amount of time for the lectin to bind cellular Pdpn thereby forming a lectin- Pdpn molecular complex comprising at least a portion of the amino acid sequence set forth in the 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]). Formation of the lectin-Pdpn molecular complex inhibits the activity of Pdpn. The method may include identifying cells over-expressed with Pdpn by immunoassaying (e g., via immunofluorescence microscopy). The tumor cells may be cancer cells, where the cancer may be, e.g., carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer.

In various aspects, a method for inhibiting cancer cell growth may be provided. The method may include identifying cells overexpressed with Pdpn and contacting the cells with an effective amount of lectin that binds sialic acid comprising 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 cell growth. ). The cells may be cancer cells, where the cancer may be. e.g.. carcinoma, leukemia, lung cancer, colon cancer. CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer. Cancer cell growth may be inhibited by about 75% as compared to a cancer cell not contacted with the lectin.

In various aspects, a pharmaceutical composition may be provided. The composition may include an isolated lectin that binds sialic acid wherein the lectin consists of the 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]), in a mixture with a pharmaceutically acceptable carrier. The composition may be formulated for parenteral administration. The composition may be formulated for oral administration.

In various aspects, a kit may be provided. The kit may include an isolated lectin that binds sialic acid wherein the lectin consists of the 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]). The kit may include at least one additional suitable component, the at least one additional suitable component being a pharmaceutically acceptable carrier, a measuring device, a buffer, a diluent, a filter, a package insert with instructions for use, or a combination thereof.

In various aspects, a chimeric molecule may be provided. The chimeric molecule may include 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 may bind sialic acid thereby reducing tumor cell growth. The polypeptide may include, e.g., a polyhistidine tag. The polypeptide may include, e.g., an immunoglobulin or a region thereof. The chimeric molecule may also include a detecting agent, such as a fluorescent moiety.

In various aspects, a method of targeting Pdpn expressed on a cell may be provided, the method including comprising the cell with a composition comprising the chimeric molecule as disclosed herein.

In various aspects, a method for reducing Pdpn-expressing cancer cell growth may be provided, the method including administering to a subject in need thereof an effective amount of the chimeric molecule as disclosed herein. The chimeric molecule may be administered parenterally. The chimeric molecule may be administered orally. The cancer may be, e.g., carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer.

In various aspects, a pharmaceutical composition may be provided. The composition may include an isolated lectin that binds sialic acid wherein the lectin consists of the 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]) in a mixture. The mixture may include a pharmaceutically acceptable material, where the pharmaceutically acceptable material is a flavor additive, a lubricant, a binder, a preservative, and/or an encapsulating material. The pharmaceutical composition may include at least one additional pharmaceutically acceptable material, where the at least one additional pharmaceutically acceptable material is a diluting agent, an artificial coloring agent, a flavor additive, a binder, a stabilizer, a natural or artificial sweetener, a thickener, a tablet-disintegrating substance, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and/or sodium carboxymethylcellulose. The pharmaceutical composition may be in a solid dosage form, and particularly may be in the form of a powder, tablet, pill, capsule, suppository, or dispersible granules. The composition may be in a liquid dosage form, and particularly may be in the form of a suspension or an emulsion.

In various aspects, a method of decreasing cartilage degradation in a subject may be provided. The method may include administering to the subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-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 a-2,3- sialic acid transmembrane glycoprotein may be Podoplanin (PDPN), which may be expressed by a chondrocyte.

In various aspects, a method of treating inflammatory joint disease (such as arthritis, such as osteoarthritis or rheumatoid arthritis) in a subject may be provided. The method may include administering to the subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-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 a-2,3-sialic acid transmembrane glycoprotein may be Podoplanin (PDPN). The total concentration of the lectin at the local level of the joint may range from, e.g., about 50 nM to about 2800 nM. Administration of the composition may reduce the baseline local level of reactive oxidative species (ROS) in the subject. The baseline local level of ROS may be reduced at least 10% compared to a normal baseline of a healthy subject. The baseline local level of ROS may be reduced by at least two-fold in the subject. The method may include administering to the subject a second active agent. The second active agent may be a nonsteroidal antiinflammatory drug (NTHE), a corticosteroid, an opiate agonist, a tumor necrosis factor (TNF) inhibitor, a disease-modifying antirheumatic drug (DMARD), or a combination thereof.

BRIEF DESCRIPTION OF FIGURES

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

Figures 1A-1B are images of SDS-PAGE results, where MASL, MAA, MALI, MAL2, and MAM (10 ug per lane) were resolved by nonreducing (1A) and reducing (IB) 12% SDS- PAGE and visualized by staining along with molecular weight markers as indicated.

Figure 2 is an image of SDS-PAGE results showing a magnified view' of MASL bands resolved by reducing SDS-PAGE with calculated molecular weights as indicated.

Figure 3 is an image of SDS-PAGE results, where shrPDPN (5 ug per lane) was resolved by reducing 12% SDS-PAGE and visualized by staining along with molecular weight markers as indicated. Figure 4A and 4B are images of Human OSCC tissue examined by Hematoxylin and eosin (H&E) stain (4 A) and immunohistochemistry (4B) to detect PDPN as indicated (bar=100 um).

Figure 5 is an image of western blotting results, where PDPN and GAPDH and expression in cells treated with 0 nM, 770 nM, 1440 nM, or 2880 nM MASL for 24 hours, with migration of molecular weight markers as indicated.

Figure 6Ais a graph illustrating the migration of cells treated with 0 nM, 770 nM. 1440 nM, or 2880 nM MASL over 24 hours, evaluated by wound healing assays (bar=100 um) with data normalized and shown as the percent of untreated controls (mean+SEM, n=3) with double and quadruple asterisks indicating p<0.01 and p<0.0001, respectively by test as indicated.

Figure 6B are images at time t=0 hours and t=24 hours for the four MASL treatments considered in Figure 6A.

Figure 7A is a graph illustrating the viability of cells treated with 0 nM, 770 nM, 1440 nM, or 2880 nM MASL for 24 hours (bar=50 um), evaluated by alamarBlue assays with data normalized and show n as the percent of untreated controls (mean+SEM, n=3) with quadruple asterisks indicating p<0.0001 by test as indicated.

Figure 7B are images at time t=24 hours of rhte four MASL treatments considered in Figure 7 A.

It should be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features illustrative of the basic principles of the invention. The specific design features of the sequence of operations as disclosed herein, including, for example, specific dimensions, orientations, locations, and shapes of various illustrated components, will be determined in part by the particular intended application and use environment. Certain features of the illustrated embodiments have been enlarged or distorted relative to others to facilitate visualization and clear understanding. In particular, thin features may be thickened, for example, for clarity or illustration.

DETAILED DESCRIPTION

The following description and drawings merely illustrate the principles of the invention. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody the principles of the invention and are included within its scope. Furthermore, all examples recited herein are principally intended expressly to be only for illustrative purposes to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor(s) to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Additionally, the term, "or," as used herein, refers to a nonexclusive or, unless otherwise indicated (e g., “or else’’ or “or in the alternative”). Also, 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.

The numerous innovative teachings of the present application will be described with particular reference to the presently preferred exemplary embodiments. However, it should be understood that this class of embodiments provides only a few examples of the many advantageous uses of the innovative teachings herein. In general, statements made in the specification of the present application do not necessarily limit any of the various claimed inventions. Moreover, some statements may apply to some inventive features but not to others. Those skilled in the art and informed by the teachings herein will realize that the invention is also applicable to various other technical areas or embodiments.

M. amurensis lectin nomenclature and composition have not been clearly defined to date. As disclosed herein, it can be seen that these lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions.

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

It is also disclosed that MASL is composed of 4 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]) having identical amino acid sequences, but unique glycosylation sites. All four isoforms included glycosylation sites on asparagines at residues 61 and 113. Asparagine was glycosylated at residue 179 on the 33 kD and 28 kD isoforms, and 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 asparagine 1 5 on the 36 kD and 33 kD isoforms.

It is also disclosed that MASL’s isoforms can serve as an agent for treating diseases that involve cells expressing the PDPN receptor, such as human oral squamous cell carcinoma (OSCC) cells. In various aspects, a MASL isoform may be utilized to treat, e.g., a cancer such as skin cancer, leukemia, lung cancer (such as non-small cell lung cancer), colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer.

In the various treatments, the lectin may be administered using any appropriate route. In some embodiments, the lectin may be administered orally. In some embodiments, the lectin may be administered nasally, buccally, rectally, or topically. In some embodiments, the lectin may be administered parenterally (for example, via subcutaneous, intravenous, intramuscular or intratumoral injection).

The lectin of the present disclosure may be isolated and optionally purified using conventional methods. For example, when isolated from its natural source, the lectin can be purified to homogeneity on appropriate immobilized carbohydrate matrices and eluted by proper haptens. See, Goldstein & Poretz (1986) In The lectins. Properties, functions and applications in biology and medicine (ed. Liener et al.), pp. 33-247. Academic Press, Orlando, Fla.; Rudiger (1993) In Glycosciences: Status and perspectives (ed. Gabi us & Gabius), pp. 415-438. Chapman and Hall, Weinheim, Germany. Alternatively, the lectin can be produced by recombinant methods according to established methods. See Streicher & Sharon (2003) Methods Enzymol. 363:47-77. As yet another alternative, lectins can be generated using standard peptide synthesis technology' or using chemical cleavage methods well-known in the art based on the amino acid sequences of known lectins or the lectin disclosed herein.

As used herein, a “subject” means a mammal, including, for example, a human, a monkey or a cat, and the like. Subjects benefiting from therapeutic treatment with lectin include subjects wherein cancer has already been identified or has started progressing or metastasizing. Subjects benefiting from prophylactic treatment with lectin include subjects with a predisposition or increased risk of cancer (e.g., because of genetics or exposure to carcinogens), wherein cancer has not yet formed, established, progressed or metastasized.

An effective amount of lectin is preferably within the range of about 0.01 mg/kg body weight to about 500 mg/kg body weight, and can be readily determined by considering the activity of the particular lectin being administered, the route of administration, the period over which the lectin is to be administered, and other factors known to those skilled in the art. Thus, the lectins described herein can be used as medicaments for the treatment of a variety of cancer pathologies by inhibiting tumor cell growth and metastasis.

The in vivo effect of a therapeutic composition may be evaluated in a suitable animal model. For example, xenogenic cancer models wherein human cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice, are appropriate in relation to cancer 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 capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

The lectin preparation, together with conventional additives, carriers or diluting agents, can be used to prepare pharmaceutical compositions, including individual doses thereof, in the form of tablets, filled capsules, or fluids such as solutions, mixtures, emulsions, elixirs or capsules filled with such, all for oral intake, as well as in the form of suppositories.

The pharmaceutical composition may be in a solid dosage form. The solid dosage form may be a powder, tablet, pill, capsule, suppository, or dispersible granule. The pharmaceutical composition may be in a liquid dosage form. The liquid dosage form may be a suspension or an emulsion.

Such pharmaceutical compositions and individual doses thereof can include conventional ingredients or principles, and such dosage forms can contain any effective concentration of the active ingredients in accordance with the intended daily dosage range. Preparations that contain approximately 100-300 mg of the lectin per individual dosage unit may be representative of an appropriate concentration. However, dosages and administration protocols for the treatment of cancers using the foregoing methods may vary with the method and the target cancer and will generally depend on a number of other factors appreciated in the art.

The pharmaceutical composition according to the invention can be administered in a wide range of dosage-forms. Carriers used to produce a pharmaceutical containing the instant lectin can include both solid and liquid substances. Solid dosage-forms may include powders, tablets, pills, capsules, suppositories, or dispersible granules. A solid carrier can be one or more substances that function as a diluting agent, flavor additive, solvent, lubricant, suspension agent, binder, preservative, tablet-disintegrating substance or encapsulating material.

In powdered form, the carrier is a finely pulverized solid including lactose, hydroxypropylmethylcellulose and PVP, mixed with an appropriate amount of finely pulverized lectin preparation.

Appropriate carriers for powder and tablet forms include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and sodium carboxymethylcellulose. The term "composition'' is meant to include, inter alia, dosage forms where the active ingredients are enclosed in an encapsulating material, any may optionally be associated with a carrier. The dosage form may including, e.g., capsules or lozenges.

Suppositories are produced by melting a low-melting point wax and distributing the lectin therein. The melted, homogeneous mixture is then poured into forms and allowed to cool.

Compositions appropriate for vaginal administration can be presented as presses, tampons, creams, gels, pastes, foams or sprays that include, in addition to the active ingredient, suitable carriers known in the art.

Compositions in liquid form include solutions, suspensions, and emulsions, for example aqueous or propylene glycol solutions, together with coloring agents, flavor additives, stabilizing agents or diluting agents as appropriate. Also included are compositions in solid form that are meant to be converted to liquid form shortly prior to consumption. These forms may include, in addition to the active ingredients, artificial colors, flavors, stabilizers, buffers, natural or artificial sweeteners, dispersing agents, thickeners, dissolving agents and the like.

For topical administration to the epidermis, the lectin composition can be presented in the form of salves, creams, gels, skin washes or transdermal plasters. Salves and creams can be formulated with an aqueous or oil base, with the addition of suitable thickeners and/or gels. Skin washes can be prepared with an aqueous or oil base and may contain one or more emulsifying agents, stabilizers, dispersing agents, thickeners or fragrances.

Compositions suitable for topical administration in the mouth include lozenges that include active ingredients in an inert, flavored base, such as sucrose and arabica gum, as well as mouth washes containing the active ingredients in a liquid carrier.

Solutions or mixtures may be administered directly to the nasal cavity using conventional means, such as drops or sprays. The composition may be produced in individual or multi-dose forms. Multi-dose forms would include a dropper, pipette or atomizer that delivers a predetermined volume of the composition.

Administration to the respiratory tract can be achieved by the use of an aerosol preparation in which the active ingredient is placed in a pressurized container together with a suitable delivery agent, such as CFC, trichlorofluormethane, dichlorofluormethane, carbon dioxide or other suitable gas. The dosage may be controlled by an appropriate valve-system.

The pharmaceutical composition is preferably provided in individual dosage units that contain a suitable amount of the active ingredient. The individual doses may be provided in a package, or as a kit that includes a measuring device, e.g.. a device for measuring oral or injectable dosages (i.e. , a measuring cup, needle, or syringe). The kit can also include, other materials such buffers, diluents, filters, and package inserts with instructions for use. A label may be present on the on the kit to indicate that the composition is used for a specific therapy, and may also indicate directions for use, such as those described above.

The compositions and methods of the present invention may also be useful in reducing the side effects of traditional chemo-radio therapies, wherein lectin is administered in conjunction with the chemo-radio therapy thereby reducing the amount of toxic dosage needed to kill cells.

In various aspects, a method for reducing tumor cell growth and migration may be provided. The method may include comprising contacting a tumor cell with an effective amount of a lectin that binds sialic acid, thereby reducing tumor cell grow th and migration. The lectin may include 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]).

In various aspects, a method for reducing tumor size and vascularization in a subject may be provided. The method may include administering to a subject in need thereof an isolated lectin that binds sialic acid, thereby decreasing tumor size and vascularization. The lectin may include an amino acid sequence as set forth in Isoform 1 ([SEQ ID NO:2J), Isoform 2 ([SEQ ID NO:3]), Isoform 3 ([SEQ ID NO:4]), or Isoform 4 ([SEQ ID NO:5]).

In various aspects, a method for treating cancer may be provided. The method may include administering to a subject in need thereof an effective amount of an isolated lectin that binds sialic acid, thereby treating the subject's cancer. The lectin may include 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]).

In various aspects, a method of targeting Pdpn expressed on a cell may be provided. The method may include administering to a subject in need thereof (e.g., a patient suffering from cancer, such as carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer) a composition comprising sufficient amounts of an isolated lectin comprising the amino acid sequence 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]). The method may include allowing sufficient amount of time for the lectin to bind cellular Pdpn, thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence 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]). The formation of the complex may inhibit the activity of Pdpn. In some embodiments, formation of the complex modifies the cells behavior as compared to cells not exposed to the lectin. In various aspects, a method of targeting Pdpn expressed on a cell may be provided. The method may include providing a cell culture comprising cells from a cancer (such as carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer) and/or normal cells. The method may include subjecting the cells to sufficient amount of an 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]). The method may include allowing sufficient amount of time for the lectin to bind cellular Pdpn thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence set forth in the 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. The method may include identifying cells over-expressed with Pdpn by immunoassaying (e.g., via immunofluorescence microscopy).

In various aspects, a method for inhibiting cell growth from a cancer (such as carcinoma, leukemia, lung cancer, colon cancer. CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer) may be provided. The method may include identifying cells overexpressed with Pdpn and contacting the cells with an effective amount of lectin that binds sialic acid, thereby reducing tumor cell growth. The lectin may include 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]). In some embodiments, cancer cell growth may be inhibited by about 75% as compared to a cancer cell not contacted with the lectin.

In various aspects, a pharmaceutical composition may be provided. The pharmaceutical composition may include an isolated lectin that binds sialic acid, in a mixture with a pharmaceutically acceptable carrier. The lectin may include the 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]).

In some embodiments, a kit may be provided. The kit may include an isolated lectin that binds sialic acid. The lectin may include the 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]). The kit may include at least one additional suitable component. The at least one additional suitable component may be a pharmaceutically acceptable carrier, a measuring device, a buffer, a diluent, a filter, a package insert with instructions for use, or a combination thereof. In various aspects, a chimeric molecule may be provided. The chimeric molecule may include 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 include 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 binds sialic acid, thereby reducing tumor cell growth. The polypeptide may include a polyhistidine tag. The polypeptide may include an immunoglobulin or a region thereof. The chimeric molecule may include a detecting agent (such as a fluorescent moiety).

In various aspects, a method of targeting Pdpn expressed on a cell may be provided. The method may include contacting the cell with a composition that includes an embodiment of the disclosed chimeric molecule.

In various aspects, a method for reducing Pdpn-expressing cancer cell growth may be provided. The method may include administering to a subject in need thereof an effective amount of an embodiment of the disclosed chimeric molecule. In some embodiments, the cancer may be carcinoma, leukemia, lung cancer, colon cancer. CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer.

In various aspects, a pharmaceutical composition may be provided. The pharmaceutical composition may be a mixture including an isolated lectin that binds sialic acid. The lectin may include the amino acid sequence as set forth in Isoform 1 ([SEQ ID NO:2]), Isoform 2 ([SEQ ID NO:3]), Isoform 3 ([SEQ ID NO:4j). or Isoform 4 ([SEQ ID NO:5]). The mixture may include a pharmaceutically acceptable material. The pharmaceutically acceptable material may be a flavor additive (e.g., an oil such as peppermint oil, a natural sweetener such as a sugar, or an artificial sweetener such as saccharin), a lubricant (for preventing adhesion to a surface, such as magnesium stearate, sodium stearyl fumarate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated vegetable oil, etc.), a binder (to hold the product together or in a particular shape, such as sugars, gelatin, gums, microcrystalline cellulose and other modified celluloses, waxes, or synthetic polymers such as polyethylene glycol, etc ), a preservative (such as benzyl alcohol, sorbic acid, methyl or propyl-p-hydroxybenzoates, etc.), and/or an encapsulating material (such as 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.

The pharmaceutical composition may include at least one additional pharmaceutically acceptable material. The at least one additional pharmaceutically acceptable material may be a diluting agent, an artificial coloring agent, a flavor additive, a binder, a stabilizer, a natural or artificial sweetener, a thickener, a tablet-disintegrating substance, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and/or sodium carboxymethylcellulose.

The pharmaceutical composition may be in a solid dosage form. The solid dosage form may be a powder, tablet, pill, capsule, suppository, or dispersible granule. The pharmaceutical composition may be in a liquid dosage form. The liquid dosage form may be a suspension or an emulsion.

In various aspects, a method of decreasing cartilage degradation in a subject may be provided. The method may include administering to the subject a pharmaceutical composition. The pharmaceutical composition may include at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-2,3-sialic acid transmembrane glycoprotein. The agent may be a lectin including 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 a-2,3-sialic acid transmembrane glycoprotein may be Podoplanin (PDPN). The PDPN may be expressed by a chondrocyte.

In various aspects, a method of treating inflammatory j oint disease (e.g., arthritis, such as osteoarthritis or rheumatoid arthritis) in a subject may be provided. The method may include administering to the subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-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 a-2,3-sialic acid transmembrane glycoprotein may be Podoplanin (PDPN). The total concentration of the lectin at the local level of the joint may be in a range from about 50 nM to about 2800 nM. In some embodiments, the administration reduces the baseline local level of reactive oxidative species (ROS) in the subject, such as reduced at least 10% compared to a normal baseline of a healthy subject. In some embodiments, the administration reduces the baseline local level of reactive oxidative species (ROS) in the subject, such as by at least two-fold in the subject. The method may include administering to the subject a second active agent, where the second active agent is a nonsteroidal anti-inflammatory drug (NTHE), a corticosteroid, an opiate agonist, a tumor necrosis factor (TNF) inhibitor, a disease-modifying antirheumatic drug (DMARD), or a combination thereof. Maackia amurensis lectins have been used in research for over 60 years. In particular, Mciackia amurensis seed lectin (MASL) has been used as an agent that binds to a-2-3-linked sialic acids. These glycosylation modifications are often found on extracellular protein receptors associated with viral infection, inflammation, and cancer [5, 6], Consequentially, MASL can target these receptors to inhibit viral infection, arthritis development, and cancer progression. For example, MASL inhibits SARS-CoV-2 spike binding to the ACE2 receptor in cell culture. MASL also targets the PDPN receptor to inhibit osteoarthritis and rheumatoid arthritis progression in mice, and suppress psoriatic epidermal hyperproliferation in skin cultures. In addition, MASL targets the PDPN receptor to inhibit melanoma cell motility and viability in culture and tumor progression in mice. MASL also targets the PDPN receptor to inhibit oral squamous cell carcinoma (OSCC) cell growth and motility and is being assessed as an oral cancer treatment in an ongoing Phase 1 human clinical trial (NCT04188665). Maackia amurensis lectin can also target receptors to inhibit non-small cell lung cancer and childhood acute lymphoblastic leukemia cell viability.

Vendors supply Maackia amurensis lectins by several different names, and the nomenclature is not clearly defined. These can be classified into two major groups: (1) MAL (M. amurensis lectin) which includes MAM (M. amurensis mitogenic), MALI, MAA (M. amurensis agglutinin), and MAA1, and (2) MAH (M. amurensis hemagglutinating) which includes MAL2, MAA, and MAA2. Notably. MAA has been included in both groups, some names including MAL and MAL 1. MAA and MAA 1 , MAH and MAL2, and MAA and M AA2 are sometimes used interchangeably. In addition, some vendors supply MAA or MAL as potentially mixed lectins without distinction.

SDS-PAGE was used to resolve MASL and other M. amurensis lectins including MAA, MALL MAL2, and MAM. MASL, MAA, MALI, MAL2, and MAM were resolved by on 12% SDS-PAGE gels (20 ug/lane) in loading buffer (2% SDS, 10% glycerol, and 0.05% bromophenol blue in 62.5 mM Tris-HCl pH6.8) with (reducing) or without (nonreducing) 10% P-mercaptoethanol, and stained with COOMASSIE® Brilliant blue R-250 (C.I. 42660) available via. e.g., Sigma-Aldrich.

Reducing gels resolved MASL, MALI, and MAM as 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 gels resolved MAA and MAL2 into smaller subunits with 2 major bands at 28 kD and 27 kD as shown in FIGS. 1A-1B.

Results from reducing SDS-PAGE (see FIG. IB) indicate that all of the M. amurensis lectins examined here contain subunits that migrate at 28 kD and 27 kD. Nonreducing SDS- PAGE (see FIG. 1A) was then employed to examine the ability of these subunits to dimerize. MASL subunits formed dimers that migrated at 72 kD, which are likely mediated by a cysteine residue in the carboxy region of the protein as previously reported. MALI and MAM subunits also produced these dimers on nonreducing gels. However, MAA and MAL2 subunits did not form dimers in these gels. Thus, these lectins represented two distinct groups with MASL, MAL 1. and MAM forming dimers, while the group containing MAA and MAL2 do not form dimers on nonreducing gels as shown in FIG. 1A.

MASL was sequenced to verify its identity. Subunits resolved at 27 kD, 28 kD, 33 kD, and 36 kD by reducing SDS-PAGE disclosed herein, and shown in FIGS. 1 A, IB, and 2. Each band was excised and examined by LC-MS/MS.

LC-MS/MS was analyzed as described in, e.g., Retzbach, et al., ‘Independent effects of Src kinase and podoplanin on 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 following modifications. 10 ug MASL was resolved by SDS-PAGE. stained with COOMASSIE® brilliant blue R-250. and bands were excised from the gels. Protein was reduced with lOmM DTT for 30 min at 60°C, alkylated with 20mM iodoacetamide for 45min at room temperature in the dark, and digested overnight with 0.2pg of either 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 chymotryptic peptides were further digested with PNGaseF. LC-MS/MS was conducted using a nano LC (DIONEX™ ULTIMATE™ 3000 RLSCnano System, ThermoFisher) interfaced with an Orbitrap Eclipse Tribrid mass spectrometer (ThermoFisher). Each sample (-25% of digests) was loaded onto a fused silica trap column (ACCLAIM™ PEPMAP™ 100 HPLC column, 75umx2cm, ThermoFisher). After washing for 5 min at 5 pl/min with 0.1 % TFA, the trap column w as brought in-line with an analytical column (Nanoease M/Z peptide BEH C18, 130A, 1.7um, 75umx250mm, Waters) for LC-MS/MS. Peptides were fractionated at 300 nL/min using a segmented linear gradient of 4- 15% B in 5min (where A: 0.2% formic acid, and B: 0.16% formic acid, 80% acetonitrile), 15- 50% B in 50min, and 50-90% B in 15min. Solution B is then returned to 4% for 5 minutes before the next run. The scan sequence began with an MSI spectrum (Orbitrap analysis, resolution 120,000, scan range from M/Z 275-1500, automatic gain control target 1E6, maximum injection time 100 ms). The top N (3 sec) duty cycle scheme was used to determine the number of MS/MS scans performed for each cycle. Precursor ions of charges 1-7 were selected for MS/MS and a dynamic exclusion of 60sec was used to avoid repeat sampling. Precursor ions were isolated in the quadrupole with an isolation window of 1.2 m/z, automatic gain control target 1E5, and fragmented with higher-energy collisional dissociation with a normalized collision energy of 30%. Fragments were scanned in Orbitrap with resolution of 15,000. MS/MS scan ranges were determined by the charge state of the parent ion but a lower limit was set to 110 m/z. Peak list MASCOT Generic Format (MGF) files were generated by Thermo Proteome Discoverer (v. 2.4) and searched against a database including relevant MASL and variant sequences plus a FASTA database composed of common lab contaminants (CRAP) using an in-house installation of GPM Fury' (X! Tandem Alanine). Mudpit searches were conducted using all mgf files from LC-MS/MS analysis of the each sample digested with different proteases. Parameters for the initial search were as follows: parent mass error ± 7 ppm, fragment mass error ± 20 ppm, and fixed modification of carbamidomethylation on cysteine and variable modifications of methionine monooxidation and asparagine deamindation. Variable modifications during three rounds of refinement were as follows: 1st round - monooxidation at methionine and tryptophan, 2nd round - deamination at glutamine and asparagine, 3rd round - dioxidation at methionine and tryptophan with protease specificity set to non-specific. Minimum 5 acceptable peptide and protein expectation scores were set at 10-2 and 10-4, respectively. Results were confirmed and analyzed by manual inspection. Evidence for O-linked glycosylation sites was not seen and N-linked sites were confirmed by PNGase F digestion.

All four MASL isoforms contain the same primary amino acid sequence. This resulting sequence was aligned with previously reported sequences. This sequence contains a cysteine at residue 272 similar to that reported by Ochoa- Alvarez et al and Yamamoto et al, as opposed to a serine in this position reported by Van Damme et al. This cysteine residue is consistent with driving dimer formation seen in nonreducing gels seen in FIG. 1 A. The MASL sequence defined here also contains threonine, serine, and glutamate 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.

The PDPN receptor has been identified as a biologically relevant MASL target. Sialic acids on PDPN are presumed to be relevant for MASL binding. Soluble recombinant human PDPN (srhPDPN) was produced in human HEK293F cells to verify sialic acid modification of this receptor.

Specifically’, a sequence encoding a protein consisting of a 19 amino acid signal peptide (SEQ ID NO:7) from mouse Ig heavy chain V region BCL1 followed by the entire extracellular region containing amino acids 23-131 (SEQ ID NO:6) of human PDPN terminating in a HIS tag (SEQ ID NO:8) was engineered into pcDNA3.1(+) and transfected into FREESTYLE™ HEK293F cells (ThermoFisher R790-07). Resulting soluble recombinant human PDPN (srhPDPN) was purified on Ni Sepharose after tangential diafiltration of the medium, followed by exchange to PBS on SUPERDEX™ 200 increase columns and analysis by SDS-PAGE. For mass spectroscopy analysis, srhPDPN (0.5 pg/pl in 25 mM ammonium bicarbonate, pH 8) was digested with either trypsin (0.01 pg/pl) or GluC (0.01 pg/pl) (Sequencing Grade; Promega) for 16 hours at 37°C. The digestion reaction was stopped by adding trifluoroacetic acid to 1%. Peptides mixtures (10 pl) were injected for LC-MS/MS analysis on an DIONEX™ ULTIMATE™ 3000 RSLCnano system in-line connected to a Q EXTRACTIVE™ HF Biopharma mass spectrometer (Thermo). Trapping was performed at 20 pl/min for 2 min in loading solvent A (0.1% trifluoroacetic in water) on a 5 mm trapping column (Thermo scientific, 300 pm internal diameter (I.D.), 5 pm beads). The peptides were separated on a 250 mm Aurora Ultimate, 1.7pm C18, 75 pm inner diameter (lonOpticks) kept at a constant temperature of 45°C. Peptides were eluted by a non-linear gradient starting at 0.5% solvent B reaching 26% solvent B (0.1% trifluoroacetic in acetonitrile) in 30 min, 44% solvent B in 38 min, followed by a 7-minute wash at 56% solvent B and re-equilibration with solvent A at a flow rate of 300 nl/min. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the 12 most abundant ion peaks per MS spectrum. Full-scan MS spectra (375-1500 m/z) were acquired at a resolution of 60,000 in the Orbitrap analyzer after accumulation to a target value of 3,000,000. The 12 most intense ions above a threshold value of 15,000 were isolated with a width of 1.5 m/z for fragmentation at a normalized collision energy' of 28% after filling the trap at a target value of 100,000 for maximum 120 ms. MS/MS spectra (200-2000 m/z) were acquired at a resolution of 15,000 in the Orbitrap analyzer. The polydimethylcyclosiloxane background ion at 445.120028 Da was used for internal calibration (lock mass) and QCloud was used to control instrument longitudinal performance during the project. Data analysis was performed with BioPharma Finder software (Thermo Fisher Scientific), using the Peptide Mapping tool. Resulting MS/MS spectra were analyzed with BioPharma Finder 3.0 software (Thermo Fisher Scientific) and mapped onto the appropriate protein sequence with trypsin or Glu-C selected as proteases. For peptide identification, the following parameters were used: maximum peptide mass of 7000 Da, mass accuracy of 5 ppm and a minimum confidence of 0.80. Deamidation of asparagine and glutamine, and oxidation of methionine and tryptophan were set as variable modifications. The search for glycosylation modifications was enabled (human specific) and the maximum number of variable modifications per peptide was set at 2.

This protein migrated between with an estimated molecular weight of 24.6 kD instead of its expected 12 kD size by SDS-PAGE as shown in FIG. 3.

This protein was sequenced by LC-MS/MS to find that PDPN was glycosylated at 26 amino acids. These glycosylation events were exclusively on serine or threonine residues. Seventeen of these 26 modifications contained sialic acid, which include glycosylation of threonine at residues 34 and 52 as previously reported.

MASL can target PDPN to inhibit human OSCC cell growth and motility. Robust PDPN expression was found on human OSCC cells by IHC of patient biopsy and Western blotting of cells adapted to culture as shown in FIGS. 4 A, 4B, and 5.

For the cell migration and viability assays, Senlbs human OSCC cells were obtained from an oral cancer patient enrolled in a clinical trial (#NCT04188665). They were maintained in DMEM (Hyclone SH30021) supplemented with 25 mM HEPES (Hyclone SH30237) and FBS (Seradigm 1400-500) at 37°C in 5% CO2 and 100% humidity and grown to confluence on 6 well tissue culture cluster plates (Falcon 353224) as described [13, 14], For migration assays, cell monolayers were scratched and visualized immediately before and 24 hours after treatment w ith 0 nM, 770 nM, 1440 nM, or 2280 nM MASL, and migration w as quantitated as the number of cells that entered 123x123 micron squares placed along the center of the w ound as described. Sister plates treated with MASL for 24 hours were incubated with alamarBlue (BioRad #BUF012A) for 4 hours and assayed (ex/em: 570/600 nm) to assess cell viability.

Western blotting was performed as described in, e.g., Hamilton, et al., '‘Effects of Maackia amurensis seed lectin (MASL) on oral squamous cell carcinoma (OSCC) gene expression and transcriptional signaling pathways” (2021), Sheehan, et al., “Heterocellular N- cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells” (2022). Confluent Senlbs cells were treated 0 nM, 770 nM, 1440 nM, or 2280 nM MASL for 24 hours, washed with PBS, transferred to microcentrifuge tubes, pelleted, aspirated, and frozen at -80°C before they were lysed in buffer (2% SDS. 10% glycerol, 10 mM EDTA, 50 nM DTT. 50 mM NaF, 0.2 mM Na3VO4, and 1 mM PMSF in 62.5 mM Tns pH 6.8). sonicated, and clarified by centrifugation. Protein (10 qg/lane) was resolved by SDS-PAGE, transferred to Immobilon-P membranes (Millipore #lPVH00010), and incubated with antisera specific for 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 mouse (JacksonlmmunoResearch #115-035-003), and rabbit (Proteintech #SA00001-2) and detected using enhanced chemiluminescence (Thermo Scientific 32209). Gels were stained with COOMASSIE® brilliant blue R-250 and membranes were stained with India ink to verify equal loading and transfer after blotting.

MASL inhibited cell motility and viability’ in a dosage dependent manner. MASL decreased cell motility by 61±8.2%, 91±2.8%, and completely at 770 nM, 1540 nM, and 3080 nM compared to untreated controls, respectively (mean±SEM, n=3) as shown in FIGS. 6A-6B. MASL also decreased cell viability^ by 29±1.5%, 48±0.3%, and 59±2.2% at 770 nM, 1540 nM. and 3080 nM compared to untreated controls, respectively as shown in FIGS. 7A-7B. However, in contrast to results from OSCC cells obtained from a different patient, MASL exposure did not decrease PDPN expression in these OSCC cells as shown in FIG. 5.

Claims

What is claimed is:

1. A method for reducing tumor cell growth and migration comprising contacting a tumor cell with an effective amount of a lectin that binds sialic acid, the lectin including 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 cell growth and migration.

2. A method for reducing tumor size and vascularization in a subject comprising administering to a subject in need thereof an isolated lectin that binds sialic acid, the isolated lectin including 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 decreasing tumor size and vascularization.

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

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

5. A method for treating cancer comprising administering to a subject in need thereof an effective amount of an isolated lectin that binds sialic acid, the isolated lectin including 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 treating the cancer.

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

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

8. A method of targeting Pdpn expressed on a cell comprising: administering to a subject in need thereof a composition comprising sufficient amounts of an isolated lectin comprising an amino acid sequence 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]); and allowing sufficient amount of time for the isolated lectin to bind cellular Pdpn thereby forming a lectin-Pdpn molecular complex comprising at least a portion of the amino acid sequence 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]): wherein formation of the lectin-Pdpn molecular complex inhibits activity of Pdpn.

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

10. The method of claim 8, wherein the subject in need thereof is suffering from cancer.

11. The method of claim 10, wherein the cancer is carcinoma, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal 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 isolated lectin is administered orally.

14. A method of targeting Pdpn expressed on a cell comprising: providing a cell culture comprising tumor cells and/or normal cells; subjecting the tumor cells to sufficient amount of an isolated lectin including 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]); and allowing sufficient amount of time for the isolated lectin to bind cellular Pdpn thereby forming a lectin-Pdpn molecular complex including 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]); wherein formation of the lectin-Pdpn molecular complex inhibits activity of Pdpn.

15. The method of claim 14. further comprising identifying cells over-expressed with Pdpn by immunoassaying.

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

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

18. A method for inhibiting cell grow th of a cancer, comprising: identifying cells overexpressed with Pdpn; and contacting the cells overexpressed with Pdpn with an effective amount of lectin that binds sialic acid, the lectin including 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 cell growth.

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, renal cancer, prostate cancer and/or breast cancer.

20. The method of claim 18. wherein cell growth of the cancer is inhibited by about 75% as compared to a cancer cell not contacted with the lectin.

21. A pharmaceutical composition comprising an isolated lectin that binds sialic acid wherein the isolated lectin includes 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]), in a mixture with a pharmaceutically acceptable carrier.

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 kit comprising: an isolated lectin that binds sialic acid wherein the isolated lectin includes 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]); and at least one additional suitable component, the at least one additional suitable component being a pharmaceutically acceptable carrier, a measuring device, a buffer, a diluent, a filter, a package insert with instructions for use, or a combination thereof.

25. A chimeric molecule comprising 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]).

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

27. The chimeric molecule of claim 25, wherein the at least one heterologous polypeptide comprises a polyhistidine 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 detecting agent.

30. The chimeric molecule of claim 29, wherein the detecting agent is a fluorescent moiety.

31. A method of targeting Pdpn expressed on a cell comprising contacting the cell with a composition comprising the chimeric molecule of claim 25.

32. A method for reducing Pdpn-expressing cell growth of a cancer, comprising administering to a subject in need thereof an effective amount of the chimeric molecule of claim 25.

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 carcinoma, leukemia, lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and/or breast cancer.

36. A pharmaceutical composition comprising an isolated lectin that binds sialic acid wherein the isolated lectin includes 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]) in a mixture.

37. The pharmaceutical composition of claim 36, wherein the mixture includes a pharmaceutically acceptable material, where the pharmaceutically acceptable material is a flavor additive, a lubricant, a binder, a preservative, and/or an encapsulating material.

38. The pharmaceutical composition of claim 36, which is in a solid dosage form, the solid dosage form being a powder, tablet, pill, capsule, suppository, or dispersible granules.

39. The pharmaceutical composition of claim 38, further comprising at least one additional pharmaceutically acceptable material, where the at least one additional pharmaceutically acceptable material is a diluting agent, an artificial coloring agent, a flavor additive, a binder, a stabilizer, a natural or artificial sweetener, a thickener, a tabletdisintegrating substance, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and/or sodium carboxymethylcellulose.

40. The pharmaceutical composition of claim 36, which is in a liquid dosage form, the liquid dosage form being a suspension or emulsion.

41. A method of decreasing cartilage degradation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-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 of claim 41, wherein the a-2,3-sialic acid transmembrane glycoprotein is Podoplanin (PDPN).

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

44. A method of treating inflammatory disease of a joint in a subject, the method comprising administering to the subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an agent that binds to a a-2,3-sialic acid transmembrane gly coprotein, 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 a-2,3-sialic acid transmembrane glycoprotein is

Podoplanin (PDPN).

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

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

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

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

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