WO2026032010A1 - Thérapie d'agrégation de protéines ciblant le glycane - Google Patents

Thérapie d'agrégation de protéines ciblant le glycane

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Publication number
WO2026032010A1
WO2026032010A1 PCT/CN2025/109763 CN2025109763W WO2026032010A1 WO 2026032010 A1 WO2026032010 A1 WO 2026032010A1 CN 2025109763 W CN2025109763 W CN 2025109763W WO 2026032010 A1 WO2026032010 A1 WO 2026032010A1
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cancer
glycan
cells
domain
lpat
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Kenward King Ho VONG
Xiao HAN
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Hong Kong University of Science and Technology
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Hong Kong University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • this invention fulfills this and other related needs. Specifically, this invention relates to the development of a novel therapeutic strategy targeting the sugar molecules present on cancer cell surface, for example, a lectin-directed protein aggregation therapy (LPAT) , which combines the strong glycan-targeting capabilities of multivalent lectins with the aggregating propensities of bacterial microcompartment proteins.
  • LPAT lectin-directed protein aggregation therapy
  • the design of this system is meant to be sensitive enough to elicit cell-specific aggregation towards invasive, metastatic tumor cells, all while being nontoxic to normal tissues.
  • the selective targeting is possible because cancer cells are known to exhibit aberrant glycosylation, with hypersialylation and hyperfucosylation being well-known examples.
  • invasive cancer cells that metastasize to other sites are also known to produce and secrete matrix metalloproteinase-9 (MMP-9) to break down the extracellular matrix.
  • MMP-9 matrix metalloproteinase-9
  • the exemplary LPAT agent of this invention is designed as a fusion construct with these main components: a glycan-targeting moiety (such as a lectin) , an aggregating protein domain, and optionally a solubilizing domain connected via a linker region that optionally contains a protease (such as MMP-9) cleavage site.
  • a glycan-targeting moiety such as a lectin
  • an aggregating protein domain such as a solubilizing domain
  • a linker region that optionally contains a protease (such as MMP-9) cleavage site.
  • MMP-9 protease
  • LPAT LPAT
  • the aim is to use it as a regularly administered drug to impair the adhesion or metastatic potential of any circulating tumor cells that may lead to cancer recurrence. Since LPAT is nontoxic in nature, it is safe to take while helping patients maintain a high quality of life that is not possible with traditional chemotherapy.
  • the present invention provides methods and compositions based on the novel concept of “cancer-activated lectin multivalency” for the purpose of specifically targeting cancer cells and inhibiting their metastatic potential.
  • the present invention provides a fusion molecule or construct comprising (1) a glycan-binding domain that specifically binds a pre-determined glycan; (2) an aggregating domain; and optionally (3) a solubilizing domain, with the solubilizing domain connected to the glycan-binding domain or the aggregating domain by a peptide linker.
  • the fusion construct is a fusion polypeptide, with the glycan-binding domain comprising a polypeptide that specifically binds the pre-determined glycan, and all domains connected by peptide bond (s) or peptide linker (s) .
  • the fusion molecule includes a non-protein portion, e.g., the glycan-targeting domain being a nucleic acid-based moiety (such as an aptamer specifically binding a pre-determined glycan) , and a protein portion, e.g., the aggregating domain and/or the solubilizing domain, with the two portions linked by a covalent bond.
  • any two of the glycan-binding domain, the aggregating domain, and the solubilizing domain are connected by a peptide linker, for example, between the solubilizing domain and the aggregating or glycan-binding domain.
  • the linker comprises a protease cleavage site, such as a cleavage site PLGLAG recognized by matrix metalloproteinase-9 (MMP-9) or matrix metallopeptidase 2 (MMP-2) .
  • the fusion construct of this invention described above and herein is a fusion protein that comprises a glycan-binding domain comprising an ACG lectin or a PSL lectin, an aggregating domain comprising a BmcH or BmcT protein unit, and the solubilizing domain comprising a small ubiquitin-like modifier (SUMO) protein or a maltose-binding protein (MBP) .
  • SUMO small ubiquitin-like modifier
  • MBP maltose-binding protein
  • the fusion protein comprises, from its N-terminus to C-terminus, the solubilizing domain, the aggregating domain, and glycan-binding domain, wherein the peptide linker connects the solubilizing domain and the aggregating domain and optionally contains a protease cleavage site: for example, the fusion polypeptide comprises, from its N-terminus to C-terminus, the MBP in the solubilizing domain, the BmcH in the aggregating domain, and the ACG lectin in the glycan-binding domain, wherein the peptide linker connects the solubilizing domain and the aggregating domain contains a protease cleavage site PLGLAG.
  • the fusion polypeptide is present in a composition, which further comprises one or more physiologically or pharmaceutically acceptable excipients.
  • the present invention provides a nucleic acid comprising a polynucleotide sequence encoding the fusion polypeptide of this invention as described above and herein.
  • an expression cassette comprising such a polynucleotide sequence operably linked to a promoter, for example, a heterologous promoter, which directs the expression of the fusion polypeptide of this invention, a vector comprising the expression cassette, as well as a host cell comprising the expression cassette or the vector.
  • the nucleic acid, the expression cassette, the vector, or the host cell is present in a composition, which further comprises one or more physiological or pharmaceutically acceptable excipients.
  • the present invention provides a method for treating cancer by suppressing the metastatic potential of cancer cells.
  • the method includes a step of administering to a subject in need thereof an effective amount of a composition comprising the fusion construct of this invention as described above and herein, including in the form of a fusion polypeptide or the nucleic acid comprising a polynucleotide coding sequence to express the fusion polypeptide.
  • the composition is administered systemically (e.g., by injection or oral ingestion) or locally (e.g., by topical application or by suppository) .
  • the composition is administered to the subject by intravenous, subcutaneous, intraperitoneal, intraosseous, intramuscular, or intratumoral injection.
  • the composition is administered orally or nasally or topically.
  • the subject is concurrently receiving chemotherapy or immunotherapy for cancer treatment, including metastatic cancer.
  • the subject is a cancer patient who previously received treatment such as surgery, chemotherapy, immunotherapy, or any combination thereof.
  • cancer cells from the subject were previously taken from the subject and analyzed to determine the presence of specific types of glycan and protease present on the cancer cell surface, for example, whether ⁇ 2,3-or ⁇ 2, 6-linked sialic acids and MMP-2 or MMP-9 might be present on the cancer cell surface.
  • the cancer being treated is breast cancer, such as metastatic breast cancer, or any other type of cancer over-expressing ⁇ 2, 3-linked sialic acid, ⁇ 2, 6-linked sialic acid, MMP-2, and/or MMP-9 on the cell surface.
  • the present invention provides a kit for treating cancer or for reducing the risk of metastasis in a cancer patient.
  • the kit includes a first container containing the composition of this invention as described above or herein, for example, comprising a glycan-targeting fusion polypeptide, and a second container containing a second anti-cancer therapeutic agent (e.g., a chemo-therapeutic agent or an immune-therapeutic agent) .
  • the kit further includes user instruction material providing description of dosing arrangements and its intended use etc.
  • Fig. 1 Design principles and strategy.
  • Fig. 1A Traditional antibodies have been ineffective against tumor-associated carbohydrate antigens (like sialic acid) as they are often found endogenously on normal tissues.
  • Fig. 1B Lectins have shown to possess potent agglutination properties when exposed to red blood cells.
  • Fig. 1C BmcH and BmcT are two important shell proteins found in bacterial microcompartments.
  • Fig. 1D The design of Lectin-directed protein aggregation therapy (LPAT) aims to exploit principles of controllable lectin multivalency, cancer-specific proteases, and targeted protein assembly to create a system sensitive enough to target invasive and hypersialylated metastatic cancer cells over normal tissues.
  • LPAT Lectin-directed protein aggregation therapy
  • Fig. 2 Design and characterization of LPAT agents.
  • Fig. 2A List of fusion proteins used in this study and their composition. Protein images were generated from the following Protein Data Bank files: MBP from 1ELJ, SUMO from 1EUV, BmcT from 5V76, BmcH from 5DJB, PSL from 3PHZ, ACG from 1WW4.
  • Fig. 2B SDS-PAGE of LPAT agents and controls 1-13 used in this study.
  • Fig. 2C Representative turbidity plot for the determination of the critical aggregation concentration of 1.
  • Fig. 2D Summary of critical aggregation concentrations obtained for LPAT agents 1-11.
  • Fig. 3 Analysis of sialic acid and MMP9 expression of various breast cancer cell lines.
  • Fig. 3A List of breast cancer cells used in this study, along with known histological subtypes and categorizations.
  • Fig. 3B Flow cytometry histograms to quantify total levels of cell surface exposed sialic acid. Cells were first treated with Ac 4 ManNAz (40 ⁇ M) , followed by labelling with DBCO-fluorescein (10 ⁇ M) . Cell suspensions were then prepared and analysed by FACS.
  • Fig. 3C Summary of FACS data to compare mean fluorescence intensities, which indirectly correlates to sialic acid expression among varying breast cancer cell lines.
  • Fig. 3C Summary of FACS data to compare mean fluorescence intensities, which indirectly correlates to sialic acid expression among varying breast cancer cell lines.
  • FIG. 3D ELISA assay done on the concentrated culture media of varying breast cancer cells to detect levels of secreted MMP9 following 1 day of incubation.
  • Fig. 4 Anti-adhesive properties of LPAT agents.
  • Fig. 4A Diagram to depict the role that P-selectins play in tethering to hypersialylated cancer cells circulating in the blood stream. This binding leads to subsequent adhesion onto the endothelium, eventually leading to the establishment of metastatic tumors.
  • Fig. 4B With multivalent binding of LPAT agents on cell surfaces, crucial sialic acid binding sites necessary for selectin-based adhesion will theoretically be occupied. This should suppress adhesion and consequently the onset and progression of metastatic tumors.
  • Fig. 4A Diagram to depict the role that P-selectins play in tethering to hypersialylated cancer cells circulating in the blood stream. This binding leads to subsequent adhesion onto the endothelium, eventually leading to the establishment of metastatic tumors.
  • Fig. 4B With multivalent binding of LPAT agents on cell surfaces, crucial sialic acid binding sites necessary for selectin-based
  • FIG. 4F Summary of the invasion assay conducted for MDA-MB-231 cells treated with LPAT agent 5 (5 ⁇ M) for 24 hr at 37°C.
  • Fig. 4G Sample images obtained for the invasion assay conducted for MDA-MB-231 cells treated with LPAT agent 5 (5 ⁇ M) for 24 hr at 37°C. The invading cells on the lower insert membranes were stained with crystal violet and imaged using a microscope at 10 ⁇ magnification. From the obtained images, the total number of invading cells was quantified and compared.
  • Fig. 4H Sample images obtained for the wound healing assay conducted for MDA-MB-231 cells treated with LPAT agent 5 (1 ⁇ M) .
  • Fig. 5 Proteomics analysis of MDA-MB-231 cells treated with MMP-9 cleavable LPAT agent 5.
  • Fig. 5A Volcano plots of differentially expressed proteins in 5-treated MDA-MB-231 cells compared to untreated MDA-MB-231 cells.
  • Fig. 5B Gene Ontology (GO) enrichment analysis of the differentially expressed proteins in 5-treated MDA-MB-231 cells. The data was categorized into molecular function, cellular components, and biological processes with the top 10 statistically most enriched terms.
  • Fig. 6 Investigation into the blood agglutinating properties of LPAT agents.
  • Fig. 6A Diagram to highlight the principle that the single glycan binding site and bulky MBP group of LPAT 5 likely prevents hemagglutination. Following MBP removal near hypersialyated and MMP-overexpressing cancer cells, oligomerization is then expected to elicit lectin multivalency that can be localized towards these cells.
  • Fig. 6A Diagram to highlight the principle that the single glycan binding site and bulky MBP group of LPAT 5 likely prevents hemagglutination. Following MBP removal near hypersialyated and MMP-overexpressing cancer cells, oligomerization is then expected to elicit lectin multivalency that can be localized towards these cells.
  • Fig. 6A Diagram to highlight the principle that the single glycan binding site and bulky MBP group of LPAT 5 likely prevents hemagglutination. Following MBP removal near hypersialy
  • Fig. 6C Hemagglutination assay run with varying concentrations of either LPAT 5 or ACG lectin 14 (1.3, 2.5, 5.0 nM) in a 1%rbc solution.
  • Fig. 6D Microscope images at 40 ⁇ magnification of a 1%rbc solution incubated with either LPAT agent 5 or ACG lectin 14 following 1 hr incubation at room temperature.
  • Fig. 7 Investigation into the anti-metastatic properties of LPAT agent 5 with a spontaneous metastasis mouse model.
  • Fig. 7A Photos of excised lungs of mice injected with 1 ⁇ 10 6 cells of either MDA-MB-231 (parental strain) or MDA-MB-231-LM2. The significantly increased tumor nodules highlight the strong metastatic properties of the MDA-MB-231-LM2 cell line.
  • Fig. 7B Comparison of total sialic acid levels as determined by metabolic labelling and analysis by FACS.
  • Fig. 7C Comparison of secreted MMP9 levels as determined by an ELISA assay done on concentrated culture media following 2 days of incubation.
  • Fig. 7D Images of whole body bioluminescence of mice from the treatment and control groups.
  • Fig. 7E Summary of quantified bioluminescent signals of mice from the treatment and control groups.
  • Fig. 7F Images of bioluminescent signals obtained from extracted lungs of mice from treatment and control groups.
  • Fig. 7G Summary of quantified bioluminescent signals obtained from extracted lungs of mice from treatment and control groups.
  • Fig. 9 Turbidity plots for the determination of critical aggregation concentrations for LPAT agents 1-11.
  • Fig. 10 Additional cell assay data on cytotoxicity, adhesion, and invasion.
  • Fig. 10A Cell viability tests were done to evaluate the cytotoxicity of LPAT agents 2-3, 4-5, 7-8, 10-11 and 12-13 against the MDA-MB-231 breast cancer cell line.
  • Fig. 10B Cell adhesion assays on plates pre-coated with extracellular matrix proteins (sourced from FBS) to determine the anti-adhesive properties of LPAT agents 2 and 3.
  • Fig. 10C Summary for the full dataset of the invasion assay conducted for MDA-MB-231 cells treated with LPAT agents 2-5 (5 ⁇ M) for 24 hr at 37°C.
  • Fig. 10A Cell viability tests were done to evaluate the cytotoxicity of LPAT agents 2-3, 4-5, 7-8, 10-11 and 12-13 against the MDA-MB-231 breast cancer cell line.
  • Fig. 10B Cell adhesion assays on plates pre-coated with extracellular matrix proteins (sourced from FBS) to determine the
  • FIG. 11 Additional cell assay data on migration.
  • Fig. 11A Summary for the full dataset of the wound healing assay conducted for MDA-MB-231 cells treated with LPAT agent 3 and 5 (1 ⁇ M) .
  • Fig. 11B All images obtained for the wound healing assay conducted for MDA-MB-231 cells treated with LPAT agent 3 and 5 (1 ⁇ M) . Images were obtained using a microscope at 5 ⁇ magnification. Effects on migration were identified by measuring changes to the wound area compared to a control (no treatment) .
  • Fig. 12 Additional proteomics analysis of MDA-MB-231 cells treated with uncleavable LPAT agent 4.
  • Fig. 12A Volcano plots of differentially expressed proteins in 4-treated MDA-MB-231 cells compared to untreated MDA-MB-231 cells.
  • Fig. 12B Gene Ontology (GO) enrichment analysis (molecular function) of the differentially expressed proteins in 4-treated MDA-MB-231 cells were plotted with the top 10 statistically most enriched terms.
  • FIG. 13 Additional microscope images at 10 ⁇ magnification for the blood agglutination assay. Chamber slides were filled with a 1%rbc solution incubated with either Fig. 13A LPAT agent 5, or Fig. 13B ACG lectin 14. Incubations were carried out for 1 hr at room temperature.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • gene means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, whereas non-naturally occurring amino acids include D-amino acids and those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. There are various known methods in the art that permit the incorporation of an unnatural amino acid derivative or analog into a polypeptide chain in a site-specific manner, see, e.g., WO 02/086075.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.
  • Polypeptide, ” “peptide, ” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • an “antibody” refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen) .
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD) .
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F (ab) ' 2, a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F (ab) ' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F (ab) ' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.
  • chimeric antibodies combine the antigen binding regions (variable regions) of an antibody from one animal with the constant regions of an antibody from another animal.
  • the antigen binding regions are derived from a non-human animal, while the constant regions are drawn from human antibodies.
  • the presence of the human constant regions reduces the likelihood that the antibody will be rejected as foreign by a human recipient.
  • "humanized" antibodies combine an even smaller portion of the non-human antibody with human components.
  • a humanized antibody comprises the hypervariable regions, or complementarity determining regions (CDR) , of a non-human antibody grafted onto the appropriate framework regions of a human antibody.
  • Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely. Both chimeric and humanized antibodies are made using recombinant techniques, which are well-known in the art (see, e.g., Jones et al. (1986) Nature 321: 522-525) .
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or antibodies synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv, a chimeric or humanized antibody) that retain the ability to specifically bind the same intended target antigen.
  • recombinant DNA methodologies e.g., single chain Fv, a chimeric or humanized antibody
  • one binding partner e.g., a pre-determined antigen
  • the first binding partner in a binding pair binds to the second binding partner in the binding pair at a level at least two times the background level and do not substantially bind in any significant amount to other molecules present in the sample.
  • specific binding to a particular glycan by a particular lectin under suitable conditions should yield at least twice of the binding signal between another glycan-lectin pair without a specific binding relationship and more typically more than 5, 10, 20, 50, or up to 100 times the non-specific binding signal.
  • An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter, especially one of a heterologous origin.
  • heterologous as used in the context of describing the relative location or position of two elements, such as two polynucleotide sequences (e.g., a promoter and a polypeptide-encoding sequence) or polypeptide sequences (e.g., a first and a second amino acid sequences in a fusion protein) , means that the two elements are not naturally found in the same relative location or position.
  • a “heterologous promoter” for a gene refers to a promoter that is not naturally operably linked to that gene.
  • heterologous polypeptide/amino acid sequence or “heterologous polynucleotide” to a protein, a fragment, or its encoding sequence is one derived from an origin other than the protein/gene.
  • cancer encompasses various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites.
  • Non-limiting examples of different types of cancer suitable for treatment using the compositions and methods of the present invention include colorectal cancer, colon cancer, anal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, rectal cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, renal cancer (e.g., renal cell carcinoma) , cancer of the central nervous system, skin cancer, oral squamous cell carcinoma, choriocarcinomas, head and neck cancers, bone cancer, osteogenic sarcomas, fibrosarcoma, neuroblastoma, glioma, mel
  • an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a control or an established comparison basis (such as the metastatic potential of an established line of cancer cells) .
  • An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100%, and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the control value.
  • a decrease is a negative change that is typically at least 10%, or at least 20%, 30%, or 50%, or even as high as at least 80%or 90%of the control value.
  • treatment includes both therapeutic and preventative measures taken to address the presence of a disease or condition or the risk of developing such disease or condition at a later time. It encompasses therapeutic or preventive measures for alleviating ongoing symptoms, inhibiting or slowing disease progression, delaying of onset of symptoms, or eliminating or reducing side-effects caused by such disease or condition.
  • a preventive measure in this context and its variations do not require 100%elimination of the occurrence of an event; rather, they refer to a suppression or reduction in the likelihood or severity of such occurrence or a delay in such occurrence.
  • an “effective amount” or a “therapeutically effective amount” means the amount of an active agent that, when administered to a subject or patient for treating a disorder, is sufficient to prevent, reduce the frequency of, or alleviate the symptoms of the disorder.
  • the effective amount will vary depending on a variety of the factors, such as a particular compound or bioactive agent used, the disease and its severity, the age, weight, and other factors of the subject to be treated. Amelioration of a symptom of a particular condition by administration of a pharmaceutical composition described herein refers to any lessening, whether permanent or temporary, that can be associated with the administration of the pharmaceutical composition.
  • a “pharmaceutically acceptable” or “pharmacologically acceptable” excipient is a substance that is not biologically harmful or otherwise undesirable, i.e., the excipient may be administered to an individual along with a bioactive agent without causing any undesirable biological effects. Neither would the excipient interact in a deleterious manner with any of the components of the composition in which it is contained.
  • excipient refers to any essentially accessory substance that may be present in the finished dosage form of the composition of this invention.
  • excipient includes vehicles, binders, disintegrants, fillers (diluents) , lubricants, glidants (flow enhancers) , compression aids, colors, sweeteners, preservatives, suspending/dispersing agents, film formers/coatings, flavors and printing inks.
  • compositions containing an active ingredient or multiple active ingredients refer to the fact that the composition does not contain other ingredients possessing any similar or relevant biological activity of the active ingredient (s) or capable of enhancing or suppressing the activity, whereas one or more inactive ingredients such as physiological or pharmaceutically acceptable excipients may be present in the composition.
  • a composition consisting essentially of a glycan-targeting fusion construct of this invention effective for reducing metastatic risk of cancer in a subject is a composition that does not contain any other agents that may have any detectable positive or negative effect on the same target process (e.g., anti-cancer metastasis efficacy) or that may increase or decrease to any measurable extent of cancer metastasis among the receiving subjects.
  • This disclosure relates to the development of a glycan-targeting therapeutic strategy, exemplified by a lectin-directed protein aggregation therapy (LPAT) , which combines the strong glycan-targeting capabilities of multivalent lectins with the aggregating propensities of bacterial microcompartment proteins.
  • LPAT lectin-directed protein aggregation therapy
  • the design is meant to create a system sensitive enough to elicit cell-specific aggregation towards invasive, metastatic tumor cells, all while being nontoxic to normal tissues.
  • LPAT agents were screened against a panel of 6 breast cancer cells lines, with the most potent agent showing preferential anti-adhesive and anti-invasive activity against the hypersialylated/MMP-9 overexpressing MDA-MB-231 cell line.
  • the present invention discloses a fusion construct that specifically targets sugar molecules (glycan) present on the surface of cancer cells, undergoes multimerization through its self-aggregating domain, and ultimately alters the cancer cell surface properties so as to inhibit the metastatic potential of the cancer cells.
  • the fusion constructs include these segments: a glycan-binding moiety that targets cancer cells over-expressing a pre-determined glycan on their surface, an aggregating moiety that allows multimerization of individual fusion construct molecules, and an optional moiety of a solubilizing protein that confers a level of solubility to the construct.
  • These moieties are typically joined through covalent bonds, such as peptide bonds, in some cases by way of peptide linkers.
  • additional amino acid residue (s) may be present not only between any two of these elements but also at the N-terminus and/or C-terminus.
  • Glycan-targeting domain this domain is responsible for the fusion construct of the present invention being able to specifically target cancer cells expressing a pre-determined glycan, such as one of the known tumor-associated carbohydrate antigens (TACA) , including truncated O-glycans, gangliosides, Lewis antigens, and polysialic acids.
  • TACA tumor-associated carbohydrate antigens
  • Any molecule that can specifically bind a relevant glycan may be used for this purpose, for example, nucleic acids such as aptamers and proteins such as lectins capable of specific binding to glycans may be used.
  • the ACG and PSL lectins can be used.
  • the ACG lectin which is derived from the fungus Agrocybe cylindracea, primarily recognizes ⁇ 2, 3-linked sialic acids.
  • the PSL lectin which is derived from the fungus Polyporus squamosus, primarily recognizes ⁇ 2, 6-linked sialic acids. Their amino acid sequences and locations within the exemplary fusion constructs of this invention are indicated in SEQ ID NOs: 1-11 and 14 (in shaded portions) .
  • amino acid sequence having a sequence identity at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to any one of the exemplary lectin sequences may be used as a part of the glycan-targeting domain for constructing the fusion polypeptide of this invention.
  • Suitable monosaccharides suitable for use to target cancer cells via a glycan-targeting domain in this invention include sialic acid, fucose, and high mannose, see, e.g., Dobie et al. Br. J. Cancer. 2021, 124 (1) , 76-90; Miyoshi et al. Biomolecules. 2012, 2 (1) , 34-45; and Boyaval et al. Cancers. 2022, 14 (6) , 1552.
  • Glycan epitopes suitable for targeting cancer cells via a glycan-targeting domain include GD2 and GD3 ganglioside, GM3 ganglioside, fucosyl-GM1 ganglioside, Globo H, Tn and sTn antigen, TF antigen, SLeA, SLeX, LeY, and polysalic acid, see, e.g., Zhe et al. Front. Cell. Dev. Biol. 2023, 11, 1076862; Ng et al. Curr. Med. Chem. 2019, 26 (16) , 2933-2947; Drivsholm et al. Ann. Oncol. 1994, 5 (7) , 623-626; Yang et al.
  • molecular moieties suitable for use as a glycan-targeting domain in this invention include glycan-binding antibodies or their fragments or variants such as nanobodies, single-chain antibodies (scFv) , DNA/RNA aptamers, and molecularly imprinted polymers, see, e.g., Yau et al. Molecules. 2015, 20 (3) , 3791-3810; Khilji et al. Cell Chem. Biol. 2022, 29 (8) , 1353-1361. e6; Lu et al. Sci. Rep. 2019, 9 (1) , 5101; D ⁇ az-Mart ⁇ nez et al. Anal. Chem. 2024, 96 (7) , 2759-2763; and Xing et al. Nat. Protoc. 2017, 12, 964-987.
  • glycan-binding antibodies or their fragments or variants such as nanobodies, single-chain antibodies (scFv) , DNA/RNA
  • Aggregating domain this domain is responsible for the aggregation of the glycan-targeting fusion construct of this invention.
  • Any molecule that has the ability to self-aggregate and form multimers can be used for producing the fusion construct of the present invention.
  • bacterial microcompartments BMCs
  • BMCs bacterial microcompartments
  • the selectively permeable shell of BMCs is made of a few thousand copies of self-assembling protein building blocks: the shell units BmcH and BmcT, and the vertex unit BmcP.
  • the hexamer-forming BmcH and trimer-forming BmcT proteins are used. Their amino acid sequences and locations within the exemplary fusion constructs of this invention are indicated in SEQ ID NOs: 1-13 (in bolded portions) .
  • An amino acid sequence having a sequence identity at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to any one of the exemplary Bmc sequences may be used as a part of the aggregating domain for constructing the fusion polypeptide of this invention.
  • molecular moieties suitable for use as an aggregating domain in this invention include helicases, ATPases, chaperon proteins, and capsid proteins, see, e.g., Sutter et al. Nat. Commun. 2021, 12(1) , 3809; Fernandez et al. Crit. Rev. Biochem. Mol. Biol. 2021, 56 (6) , 621-639; Zhao et al. Nat. Commun. 2021, 12 (1) , 6439; Wang et al. Nature. 2011, 471 (7338) , 331-335; and Pornillos et al. Cell. 2009, 137 (7) , 1282-1292.
  • Solubilizing domain this domain is responsible for conveying a desired level of solubility to the glycan-targeting fusion construct of this invention.
  • Any molecule, especially protein, that provides a significant degree of solubility may be used for this purpose.
  • the ⁇ 12 kDa SUMO protein and the ⁇ 43 kDa maltose-binding protein (MBP) may be used.
  • Their amino acid sequences and locations within the exemplary fusion constructs of this invention are indicated in SEQ ID NOs: 2-5, 7, 8, and 10-13 (in underlined portions) .
  • amino acid sequence having a sequence identity at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to any one of the exemplary SUMO or MBP sequences may be used as a part of the solubilizing domain for constructing the fusion polypeptide of this invention.
  • molecular moieties suitable for use as a solubilizing domain in this invention include NusA, glutathione S-transferase (GST) , and TrxA proteins, see, e.g., Fox et al. FEBS Lett. 2003, 537 (1-3) , 53-57; Marblestone et al. Protein Sci. 2006, 15, 182-189; Nallamsetty et al. Protein Expr. Purif. 2006, 45 (1) , 175-182; et al. Methods Enzymol. 2015, 559, 127-139; and Yasukawa et al. J. Biol. Chem. 1995, 270 (43) , 25328-25331.
  • Linkers the glycan-targeting domain, the aggregating domain, and the optional solubilizing domain of the fusion construct of this invention are connected via covalent bonds, e.g., peptide bonds, and in some cases involving the use of peptide linkers, especially when the fusion construct is a fusion protein.
  • a suitable linker may be as few as 1 to 2 amino acids and as many as 30 to 40 or up to 50 amino acids in length. For instance, it may be between 2 to 30, 3 to 30, 2 to 25, 3 to 25, 2 to 20, 3 to 20, 5 to 25, 5 to 20, 5 to 15, 7 to 20, 8 to 20, 7 to 15, 8 to 15, 7 to 10, or 8 to 10 amino acids in length.
  • a protease cleavage site may be included in a linker, for example, to allow release of the solubilizing domain from the fusion construct upon contact with targeted cancer cell surface where the pertinent protease (e.g., MMP-9 or MMP-2) is present and especially over-expressed.
  • additional amino acids in the same length range may be included at the N-terminus, the C-terminus, or both of the fusion polypeptide.
  • cancer cell surface expressed enzymes suitable for use to create a cleavage site in a cancer-responsive linker in this invention include ADAM10/ADAM17, Meprin ⁇ /Meprin ⁇ , kallikreins (KLKs) , and cathepsins, see, e.g., Jiang et al. BMC Cancer, 2021, 21, 149; Tsang et al. EBioMedicine. 2018, 38, 89-99; Lottaz et al. PLoS One. 2011, 6 (11) , e26450; Avgeris et al. Biol Chem. 2012, 393 (5) , 301-317; and Tan et al. World J. Biol. Chem. 2013, 4 (4) , 91-101.
  • the activity of the fusion construct to alter cancer cell surface properties and inhibit metastatic potential can be tested and confirmed in a cell migration assay known in the field and disclosed herein. Briefly, a standard wound healing assay is performed a line of suitable cancer cells (e.g., MDA-MB-231) is used in to measure changes in the ability of the cells to close a wound gap in the presence or absence of a candidate agent. An inhibitory effect is deemed present when a decrease in the wound closure rate of the cells incubated in the agent for a given time period (e.g., 18 hours) is observed, and the agent is determined as effective for suppressing cancer metastatic potential.
  • a given time period e.g. 18 hours
  • cancer cells having a certain glycan or protease expression profile on the cell surface are most effectively treated with the methods and compositions of this invention, for example, breast cancer cells expressing at least 1/5, or at least 1/3, preferably more, of the level of a pertinent glycan or protease, corresponding to the level of the glycan or protease expressed on the MDA-MB-231 cell surface.
  • cancer cells from a patient may be pre-screened for glycan/protease expression profile to determine whether a fusion construct of this invention targeting the corresponding glycan and/or cleavable by the corresponding protease would provide an effective means for treating or preventing cancer metastasis.
  • cancer cells suitable for this glycan-targeting aggregation therapy express on their surface an increased level of a glycan and/or protease, e.g., of at least 2, 3, 4, 5, or 10 times or more, compared to the non-cancerous cells of the same tissue type.
  • the individual elements of the glycan-targeting aggregation construct of this invention have known amino acid sequences, see, e.g., Figure 8 and SEQ ID NOs: 1-14.
  • the construct be chemically synthesized using conventional peptide synthesis or other protocols well known in the art.
  • Polypeptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963) ; Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds. ) , Academic Press, N. Y., vol. 2, pp. 3-284 (1980) ; and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984) .
  • N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e., polystyrene beads.
  • the peptides are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxy group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N- ⁇ -protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4- ( ⁇ - [2, 4-dimethoxyphenyl] -Fmoc-aminomethyl) phenoxy resin
  • tert-alkyloxycarbonyl-hydrazidated resins such as 4- ( ⁇ - [2, 4-dimethoxyphenyl] -Fmoc-aminomethyl) phenoxy resin
  • tert-alkyloxycarbonyl-hydrazidated resins and the like.
  • the C-terminal N- ⁇ -protected amino acid is first attached to the solid support.
  • the N- ⁇ -protecting group is then removed.
  • the deprotected ⁇ -amino group is coupled to the activated ⁇ -carboxylate group of the next N- ⁇ -protected amino acid.
  • the process is repeated until the desired peptide is synthesized.
  • the resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments.
  • the glycan-targeting aggregation agent of this invention can be produced using routine techniques in the field of recombinant genetics, relying on the polynucleotide sequences encoding the polypeptide disclosed herein.
  • a strong promoter to direct transcription e.g., in Sambrook and Russell, supra, and Ausubel et al., supra.
  • Bacterial expression systems for expressing the polypeptide are available in, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available.
  • the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. In some cases, an inducible promoter is preferred.
  • Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a recombinant polypeptide, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989) ; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990) ; Morrison, J. Bact. 132: 349-351 (1977) ; Clark-Curtiss &Curtiss, Methods in Enzymology 101: 347-362 (Wu et al., eds, 1983) ) .
  • the present invention also provides pharmaceutical compositions or physiological compositions comprising an effective amount of a glycan-targeting fusion construct that inhibits the metastatic potential of cancer cells overexpressing a particular glycan targeted by the fusion construct, such as one of the LPAT agents described herein.
  • Such compositions also include one or more pharmaceutically or physiologically acceptable excipients or carriers.
  • Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) . For a brief review of delivery methods, see, Langer, Science 249: 1527-1533 (1990) .
  • the pharmaceutical compositions of the present invention can be administered by various routes, e.g., oral, nasal, subcutaneous, transdermal, intramuscular, intravenous, or intraperitoneal.
  • the preferred routes of administering the pharmaceutical compositions are local delivery to an organ or tissue affected by cancer, especially one with metastatic potential (e.g., intratumoral injection to a tumor) at daily doses of about 0.01 -5000 mg, preferably 5-500 mg or 10-250 mg, for example, 20-100 mg, of a glycan-targeting fusion construct of this invention for a 70 kg adult human per day.
  • the appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.
  • inert and pharmaceutically acceptable carriers are used.
  • the pharmaceutical carrier can be either solid or liquid.
  • Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
  • a solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
  • the carrier is generally a finely divided solid that is in a mixture with the finely divided active component, e.g., a glycan-targeting fusion construct of the present invention.
  • the active ingredient e.g., a glycan-targeting fusion construct
  • the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.
  • Powders and tablets preferably contain between about 5%to about 70%by weight of the active ingredient (such as a glycan-targeting fusion construct) .
  • suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.
  • compositions can include the formulation of the active compound of a glycan-targeting fusion construct with encapsulating material as a carrier providing a capsule in which the fusion construct (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the fusion construct.
  • a carrier providing a capsule in which the fusion construct (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the fusion construct.
  • cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
  • Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration.
  • Sterile water solutions of the active component e.g., a glycan-targeting fusion construct of this invention
  • sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • Sterile solutions can be prepared by dissolving the active component (e.g., a glycan-targeting fusion construct of this invention) in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile component in a previously sterilized solvent under sterile conditions.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more preferably from 5 to 9, and most preferably from 7 to 8.
  • compositions containing a glycan-targeting fusion construct of this invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a metastatic cancer in an amount sufficient to prevent, cure, reverse, or at least partially slow or arrest the symptoms of the condition and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.
  • Amounts effective for this use will depend on the severity of the condition and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of the glycan-targeting fusion construct per day for a 70 kg patient, with dosages of from about 5 mg to about 500 mg of the fusion construct per day for a 70 kg patient being more commonly used.
  • compositions containing glycan-targeting fusion construct of this invention are administered to a patient who has been diagnosed with cancer and is at risk of developing metastasis in an amount sufficient to delay or prevent metastasis.
  • an amount is defined to be a "prophylactically effective dose. "
  • the precise amounts of the fusion construct again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 2,000 mg of the fusion construct for a 70 kg patient per day, more commonly from about 5 mg to about 500 mg for a 70 kg patient per day.
  • the pharmaceutical formulations should provide a quantity of a glycan-targeting fusion construct of this invention sufficient to effectively inhibit cancer metastasis in the patient, either therapeutically or prophylactically.
  • the patient has already been given another form of cancer treatment (e.g., surgery, chemotherapy, immunotherapy, or any combination thereof) at least, e.g., about 1, 2, 3, or 4 weeks prior, or is currently receiving such cancer treatment, or is scheduled to start receiving such cancer treatment shortly, for example, within the next 1, 2, 3, or 4 weeks.
  • another form of cancer treatment e.g., surgery, chemotherapy, immunotherapy, or any combination thereof
  • a cancer patient may be administered an effective amount of a glycan-targeting fusion construct described here, as deemed appropriate by an attending physician, along with another anti-cancer therapeutic agent known to be effective for suppressing cancer cell proliferation as a means of intervention therapeutically or prophylactically.
  • a glycan-targeting fusion construct described here as deemed appropriate by an attending physician
  • another anti-cancer therapeutic agent known to be effective for suppressing cancer cell proliferation as a means of intervention therapeutically or prophylactically.
  • various treatment strategies are available for treating cancer (especially solid cancer) in these patients including but not limited to, surgery, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy, or any combination thereof.
  • a glycan-targeting fusion construct of the present invention may be used concurrently, shortly before or after, together with one or more of these therapies.
  • one or more of these previously known effective anti-cancer therapeutic agents can be administered to subjects in need of treatment.
  • the active agents including the glycan-targeting fusion construct
  • the active agents may be administered concurrently each in an effective amount, either together in a single composition or separately in two or more separate compositions.
  • chemotherapeutic agents are known to be effective for use to treat various cancers.
  • a “chemotherapeutic agent” encompasses any chemical compound exhibiting suppressive effect against cancer cells, thus useful in the treatment of cancer.
  • Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs) , anti-progesterones, estrogen receptor down-regulators (ERDs) , estrogen receptor antagonists, leutinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and anti-sense oligonucleotides that inhibit expression of genes implicated in abnormal cell proliferation or tumor growth.
  • anti-cancer therapeutic agents include alkylating agents such as altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, and uramustine; anthracyclines such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, and zor
  • Immunotherapeutic approaches useful for cancer treatment include (1) active immunotherapy, which directs the immune system to specifically target the cancer cells, e.g., targeted antibody therapy and cell-based immunotherapy such as CAR T cell therapy; and (2) passive immunotherapy, e.g., using checkpoint inhibitors and cytokines to stimulate the immune system without specifically targeting cancer cells.
  • active immunotherapy which directs the immune system to specifically target the cancer cells
  • cell-based immunotherapy such as CAR T cell therapy
  • passive immunotherapy e.g., using checkpoint inhibitors and cytokines to stimulate the immune system without specifically targeting cancer cells.
  • Various monoclonal antibodies are used in targeted antibody therapy.
  • antibodies and their conjugates include adotrastuzumab (HER2) , alemtuzumab (CD52) , bevaclzumab (VEGF) , brentuximab (CD30) , capromab (PSMA) , cetuximab (EGFR) , elotuzumab (SLAMF7) , ibritumomab (CD20) , necitumumab (EGFR) , obinutumab (CD20) , ofatumumab (CD20) , olaratumab (PDGFRA) , panitumumab (EGFR) , pertuzumab (HER2) , ramucirumab (VEGFR2) , rituximab (CD-20) , trastuzumab (HER-2) , inotuzumab-ozogamicin (CD22) , gemtuzumab-ozogamicin (CD33)
  • CTLA4 ipilimumab
  • PD-1 PD-1
  • PD-L1 ipilimumab
  • Cytokines for use in the treatment of cancer and associated conditions include granulocyte colony-stimulating factor (G-CSF) , granulocyte macrophage colony-stimulating factor (GM-CSF) , interleukin-2 (IL-2) , and interleukin-11 (IL-11) .
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • IL-2 interleukin-2
  • IL-11 interleukin-11
  • the invention also provides compositions and kits for practicing the methods described herein to treat cancer, especially for inhibiting cancer metastasis, by administering a glycan-targeting fusion construct of this invention to a cancer patient.
  • a cancer patient with or without a metastasis diagnosis may be treated.
  • Kits for the therapeutic use of a glycan-targeting fusion construct of this invention typically include one container containing a composition comprising the glycan-targeting fusion construct (such as one of the LPAT agents shown in Figure 8) .
  • a composition comprising the glycan-targeting fusion construct (such as one of the LPAT agents shown in Figure 8) .
  • such composition is formulated for delivering the glycan-targeting fusion construct, e.g., by injection such as subcutaneous, intravenous, intramuscular, intraperitoneal, or intratumoral.
  • the kit includes at least one, possibly two or more anti-cancer therapeutic agents, known for their effectiveness in treating cancer, for example, any one or more of the therapeutic agents known/used in the medical field or described herein, or which might belong to any of the following 3 categories: (A) chemotherapeutic drugs, e.g., drugs capable of killing or suppressing cells that are actively undergoing proliferation; (B) immunotherapeutic agents, e.g., monoclonal antibodies for targeted antibody therapy, checkpoint inhibitors, and cytokines; and (C) cell-based therapeutic agents, e.g., those used in CAR T cell therapy or other immune cell therapy.
  • chemotherapeutic drugs e.g., drugs capable of killing or suppressing cells that are actively undergoing proliferation
  • immunotherapeutic agents e.g., monoclonal antibodies for targeted antibody therapy, checkpoint inhibitors, and cytokines
  • C cell-based therapeutic agents, e.g., those used in CAR T cell therapy or other immune cell therapy.
  • kits of this invention may provide instruction manuals to guide users in the proper administration of the composition comprising the glycan-targeting fusion construct of this invention, optionally in combination with one or more anti-cancer therapeutic agents, to a subject deemed in need of such treatment by a physician (e.g., a cancer patient whose cancer may or may not have metastasized) , the schedule (e.g., dose and frequency of administration) and route of administration, and the like.
  • a physician e.g., a cancer patient whose cancer may or may not have metastasized
  • the schedule e.g., dose and frequency of administration
  • TACA tumor-associated carbohydrate antigen
  • sialic acids which are also referred to as N-acetylneuraminic acids. These 9-carbon carboxylated monosaccharides are typically found in animal tissues and fluids as parts of varying glycoproteins and glycolipids. With an increase in sialic acid levels (hypersialylation) on cell surfaces, researchers quickly identified this phenomena as a consistent occurence for many cancer types (i.e., 40–60%through enhanced immune evasion and migration. 6, 7
  • TACA-targeting antibodies are still principally composed from glycans of human-origin.
  • the neurotoxicity of GD2-targeting dinutuximab and naxitamab is attributed to the fact that healthy cells also express GD2. 12
  • the selectivity of TACA-targeting antibodies is highly dependent on dosages that can operate within a narrow therapeutic threshold range. Otherwise, normal cells expressing endogenous levels of targeted glycans are likely to be affected by antibody off-targeting and the consequent adverse side effects (Figure 1A) .
  • lectins also referred to as carbohydrate binding proteins, this large family of proteins are known to bind varying glycan ligands in a specific and reversible nature. 13 Prominant examples of their applications in targeted therapeutics can be seen with the development of lectin-drug conjugates, 14-18 and lectin-conjugated nanoparticles. 19-23
  • multivalent lectins promote clumping of red blood cells in a process referred to as agglutination ( Figure 1B) . 24 These cell clumps eventually block blood vessels throughout the body, depriving tissues of oxygen and nutrients. Due to the risks of blood agglutination, no lectin-based targeted therapeutics have ever progressed past clinical trials.
  • the primary aim of this study is to introduce an alternative strategy for glycan targeting that can circumvent issues related to both traditional antibody-and lectin-based therapies.
  • a concept referred to as “cancer-activated lectin multivalency” will be pursued.
  • the goal of such system expects that binding to normal cells (basal levels of glycosylation) will occur, but lectin multivalency can only be activated in the presence of highly metastatic cancer cells (hyperglycosylated surfaces) to elicit a biological effect. In this manner, cell selectivity should be improved while also avoiding any risk of blood agglutination.
  • targeted peptide assemblies is an approach that aims to have soluble precursor peptides accumulate to specific cells, where a trigger event can lead to aggregation/gelation.
  • precursor targeting is typically aided by directed groups, 27 or reliant on activation by tumor-associated enzymes.
  • 28 Using this approach, studies focused on the aggregation/gelation of peptide nanoassemblies have been done with much success. 29 With consideration that the low molecular weight of peptide precursors often requires higher dosages (low to high millimolar range) to reach critical aggregation concentrations, we were inspired to find a protein-based aggregating biomolecule that could potentially operate at lower concentrations to improve sensitivity.
  • BMCs Bacterial microcompartments
  • Figure 1C specific metabolic processes
  • the selectively permeable shell of BMCs is made of a few thousand copies of self-assembling protein building blocks: the shell units BmcH and BmcT, and the vertex unit BmcP.
  • the most abundant shell unit is the BmcH protein, which forms a cyclic homohexamer that self-assembles to form an impermeable wall.
  • the BmcT shell protein exists to form a cyclic trimer (pseudohexamer) with a central pore opening.
  • BMCs display remarkable self-assembling properties, researchers have looked to exploit them for varying bioengineering applications. For example, the Kerfeld group were able to develop unique BMC architectures by simply creating protease-cleavable SUMO-BmcH fusions. In doing so, the natural self-assembling properties could be thwarted during expression/purification, allowing these proteins to form unnatural BmcH-based nanotubes upon controlled protease exposure.
  • lectin multivalency defined as the collective binding strength of multiple glycan ligands to multiple lectin receptors, is not just limited to nature. Numerous studies have created varying bioinspired materials to possess “engineered multivalency. ” For example, the Hudalla group used ⁇ -helical coiled-coil domains to create trimeric galectin-3 assemblies capable of tissue-specific enzyme activity. 34 In another study, the Turnbull group also exploited coiled-coil domains by appending them to one face of the GM1 ganglioside-targeting cholera toxin to induce supramolecular assembly. 35 And finally, multivalency control can also be manipulated via a connected glycan network. This was shown by the Tanaka group, whom have decorated albumin with multiple complex N-glycans to influence its in vivo distribution for targeted drug release/synthesis. 36-40
  • LPAT lectin-directed protein aggregation therapy
  • LPAT agents 1-13 are briefly summarized on Figure 2A and detailed in Figure 8.
  • ACG derived from the fungus Agrocybe cylindracea
  • PSL derived from the fungus Polyporus squamosus
  • hexamer-forming BmcH and trimer-forming BmcT were chosen.
  • the solubilizing unit the ⁇ 12 kDa SUMO protein and the ⁇ 43 kDa maltose-binding protein (MBP) were chosen.
  • SUMO was chosen on the basis of its previous use in preventing BmcH assembly during purification.
  • 33 MBP is a well-known protein capable of increasing the water solubility of its fusion partners.
  • the PLGLAG linker was chosen due its known cleavage activity by matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9) .
  • both MMP-2 and MMP-9 are enzymes known to be overexpressed in cancer cells to facilitate invasion and tumor metastasis.
  • 48, 49 In cases where MMP cleavage is not desired, the random sequence GNGFVG was used as a non-cleavable linker. All protein complexes used in this study were confirmed by size on SDS-PAGE ( Figure 2B) .
  • aggregation assay was first carried out. Protein aggregates in solution are known to scatter incoming light, 50 so turbidity at 340nm was used to quantify aggregation. To perform this assay, all proteins were purified in urea-supplemented buffers to ensure protein denaturation. Serial dilutions made of these protein solutions were then individually dialyzed in urea-free buffer. With the gradual elimination of urea, refolded protein complexes are then sensitive to BmcH-facilitated self-assembly and concentration-dependent aggregation. From this assay, only LPAT agents 1, 6, and 9 showed any visible protein aggregation.
  • TNBC triple negative breast cancers
  • MDA-MB-231 basal B cell lines like MDA-MB-231 and Hs578t are identified as more invasive and have stem/progenitor-like characteristics. This fact serves to justify why MDA-MB-231 possesses such high levels of MMP9.
  • Figure 3E A summary chart of the relative balance between sialic acid expression and cell surface MMP2/9 activities are depicted on Figure 3E. With consideration of this data, MDA-MB-231 was naturally chosen as the model cell line to screen LPAT agents developed in this study.
  • hypersialylated cancer cells circulating in the blood stream will initially tether with P-selectin, causing the cells to roll and eventually adhere onto the endothelium. Subsequent extravasation then allows these invasive cells to establish metastatic tumors.
  • LPAT agents 2 and 3 gave a ⁇ 54%difference while agents 4 and 5 gave a ⁇ 36%difference.
  • the proteins 12 and 13 were also tested and showed no activity. All other LPAT pairs (7 and 8, 10 and 11) did not show significant differences between uncleavable/cleavable proteins, nor the overall ability to impair cell adhesion.
  • MBP-containing LPAT agents 4 and 5 were screened against a panel of 6 breast cancer cell lines for their ability to impair cell adhesion (Figure 4D) .
  • Figure 4D MBP-containing LPAT agents 4 and 5 were screened against a panel of 6 breast cancer cell lines for their ability to impair cell adhesion.
  • Figure 10B Similar results were also observed when utilizing SUMO-containing 2 and 3 ( Figure 10B) .
  • the next assay analyzed the inhibitory effects of LPAT agent 5 on cell migration. This was carried out using a standard wound healing assay ( Figures 4H-I, Figures 11A-B) , which measures the ability of MDA-MB-231 cells to close a wound gap under varying conditions. When incubated with agent 5, an inhibitory effect was observed where the wound closure rate in 18 hours could be suppressed by up to 63%.
  • LPAT agents will need to display non-existent or highly depressed blood agglutination properties.
  • one common adjustment is to simply decrease the number of binding sites on multivalent lectins is through genetic modifications. 71 Doing so, however, adversely affects in vivo avidity.
  • LPAT agent 5 acts as a precursor that possesses only one glycan binding site.
  • agent 5 is not expected to agglutinate red blood cells ( Figure 6A) .
  • Figure 6A Once MMP-dependent cleavage of the solubilizing unit is induced near cancer cells, however, BmcH oligomerization can create a hexamer complex possessing strong lectin multivalency.
  • LPAT agents should not be able to form multivalent complexes.
  • 72 LPAT agent 5 and a control (ACG lectin 14) were incubated in a 1%red blood cells suspension and then allowed to stand in v-bottom 96-well plates ( Figure 6C) .
  • Figure 6C v-bottom 96-well plates
  • MDA-MB-231-LM2 is also encoded with firefly luciferase, which makes in vivo tumor detection possible through bioluminescent imaging.
  • mice were then injected into 5-week-old female nude mice, which were then arranged into control and treatment groups. While the control group received a saline solution, the treatment group received a dosage of 12 mg/kg of LPAT 5. All solutions were administered via intravenous tail vein injections. Following a period of 20 weeks, mice were then imaged for tumor burden via in vivo bioluminescent imaging (Figure 7D) . From this data, a clear depreciation of tumor burden in the upper body region of mice can be seen with the treatment group when compared to the control ( Figure 7E) . To consolidate this data, all mice were then sacrificed, and their lungs were excised and imaged ( Figure 7F) . Quantification of lung tumor burden by bioluminescent imaging again showed a clear reduction of tumor burden with the treatment group compared to the control ( Figure 7G) . CONCLUSION
  • any potential aftercare drugs would need to fulfill two main requirements: 1) to be selective enough to only target highly metastatic cancer cells, and 2) to be safe and not cause any adverse health effects typically associated with cancer treatment.
  • the design of LPAT fusion proteins was done with these two requirements in mind.
  • the targeting of LPAT is meant to be dually dependent on two important characteristics of metastatic breast cancer cells.
  • the first barrier is the need for metastatic cells to be hypersialylated. In this manner, LPAT agents will not only disrupt selectin-based adhesion, 64, 65 but should also suppress immune evasion of hypersialylated cells caused by Siglec-sialic acid inhibitory immune interactions. 6, 7
  • the second barrier is the need for metastatic cells to overexpress MMP2/9. This is a key factor in combating invasive breast cancer cells as studies show that increased levels of MMP-2 and MMP-9 confer a higher risk towards distant and lymph node metastases. 53
  • LPAT agents are non-toxic and do not possess any hemagglutination properties that are typically associated with lectins.
  • LPAT fusion proteins were synthesized by Genscript and inserted into pET-21a (+) vectors between differing combinations of NdeI, BamHI, HindIII, and XhoI cut sites. LPAT protein sequences are shown in Figure 7.
  • plasmids were transformed into One Shot TM BL21 (DE3) Chemically Competent E. coli (ThermoFisher) and then incubated on Luria-Bertani (LB) Agar plates with ampicillin (50 ⁇ g/mL) overnight at 37 °C.
  • Isolated colonies were picked and cultured in 7 mL LB broth with ampicillin (50 ⁇ g/mL) overnight in shaking incubators at 37 °C. These overnight cultures were then used to inoculate larger LB cultures (500 mL) , which were grown in shaking incubators at 37 °C until an O. D. reading (at 600 nm) of 0.6 was reached.
  • O. D. reading at 600 nm
  • cultures were supplemented with 0.5 mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) and then grown for an additional 4 hours at 26 °C.
  • Bacterial pellets were obtained through centrifugation (7,350 rpm at 4 °C for 10 min) and then resuspended in lysis buffer (20 mM Tris, 300 mM NaCl, 1 mM PMSF, pH 7.4) supplemented with a Pierce protease inhibitor tablet (ThermoFisher) . Sonication was performed (5s on/10s off for 15 min) , followed by centrifugation (12,000 rpm at 4 °C for 20 min) to isolate the supernatant. LPAT protein purification was carried out using affinity chromatography columns connected to an start FPLC system (Cytiva) .
  • the supernatant was loaded onto a HisPur Ni-NTA Cartridge (ThermoFisher) and washed with at least 10 column volumes of an equilibration buffer (20 mM Tris, 300 mM NaCl, pH 7.4) .
  • An imidazole gradient (0–300 mM) was then applied to the column by mixing the equilibration buffer with an elution buffer (20 mM Tris, 300 mM NaCl, 300 mM imidazole, pH 7.4) .
  • the supernatant was loaded onto a MBPTrap HP Cartridges (Cytiva) and washed with at least 10 column volumes of an equilibration buffer (20 mM Tris, 200 mM NaCl, pH 7.4) .
  • a maltose gradient (0–10 mM) was then applied to the column by mixing the equilibration buffer with an elution buffer (20 mM Tris, 200 mM NaCl, 10 mM maltose, pH 7.4) .
  • Eluted protein fractions were analyzed by SDS-PAGE, with appropriate fractions then collected and combined.
  • Approximate protein sizes were obtained by comparison to BSA (66 kDa) and a Gel Filtration Standard (Bio-rad) that contains thyroglobulin, ⁇ -globulin, ovalbumin, and myoglobin (17–670 kDa) .
  • urea was supplemented into the equilibration (20 mM Tris, 300 mM NaCl, 6 M urea, pH 7.4) and elution (20 mM Tris, 300 mM NaCl, 300 mM imidazole, 6 M urea, pH 7.4) buffers to promote protein unfolding. Following volume reduction using 30K MWCO protein concentrators and determination of protein concentration, serial dilutions of each protein was prepared. Protein solutions of varying concentrations were then placed into SnakeSkin Dialysis Tubing (ThermoFisher) and dialyzed twice against 3L of 10 mM phosphate buffer.
  • ThermoFisher SnakeSkin Dialysis Tubing
  • This step allows the removal of urea, thereby allowing the protein to refold when incubated at 37 °C in 96-well UV transparent plates (Beyotime) .
  • absorbance values at 340 nm were obtained using a VANTAstar Microplate Reader (BMG) . Readings were plotted on a logarithmic scale using GraphPad Prism 9 software.
  • MDA-MB-468, HCC1937, Hs578t, and T47D were obtained from the iCell Bioscience (China)
  • MCF-7 and MDA-MB-231 were obtained from ATCC (USA) via donation from Prof. Randy YC Poon.
  • MDA-MB-231-LM2 was obtained from Prof. Joan Massagué via an MTA with Memorial Sloan Kettering Cancer Center, New York.
  • MDA-MB-468, MDA-MB-231, Hs578t, and T47D were maintained in Dulbecco’s modified Eagle media (DMEM; Gibco)
  • HCC1937 and MCF-7 were maintained in RPMI media (Gibco) .
  • FBS heat-inactivated fetal bovine serum
  • P/S penicillin-streptomycin
  • ELISA assay To quantify and compare secreted MMP-9 among the various breast cancer cell lines, a human MMP-9 ELISA kit (Excell Bio) was used according to the manufacturer’s protocol. 2 ⁇ 10 6 of each cell line was first seeded onto a 10 cm tissue culture plate (4 ⁇ ) and incubated overnight at 37 °C. The growth media was then replaced with 10 ml of serum-free growth media and incubated for an addition 2 days at 37 °C. The growth media from plates of each cell line were then collected and concentrated to a volume of 330 ⁇ l. To begin the ELISA analysis, 100 ⁇ l of the concentrated growth media was loaded into each well of a 96-well microplate pre-coated with the capture antibody for 90 min.
  • analyte levels were measured by the addition of tetramethylbenzidine to each well. Absorbance measurements were read at 450 nm using a VANTAstar Microplate Reader (BMG) and analyte levels were then extrapolated from a standard curve.
  • BMG VANTAstar Microplate Reader
  • Cytotoxicity assays Cell viability was determined using a colorimetric MTS Assay Kit (Abcam) . Cells were first seeded onto 96-well plates at a density of 1 ⁇ 10 4 cells per well and grown overnight at 37 °C. The media was then removed, followed by the incubation of various concentrations of LPAT proteins used in this study. In general, 20 ⁇ l of LPAT proteins were added to 80 ⁇ l of growth media. Following an incubation time of 1 day, the media was removed and replacing with 20 ⁇ l of MTS reagent and 80 ⁇ l of growth media.
  • Adhesion assay using FBS-coated plates The 96-well assay plates were first precoated overnight with extracellular matrix proteins from FBS (growth media + 10%FBS) . Separate 6-well plates were seeded at a density of 3 ⁇ 10 5 cells per well and grown overnight at 37 °C. After media removal, cells were then incubated with fresh growth media supplemented with varying proteins concentrations (5 ⁇ M) . In general, 25 ⁇ l of protein stock solution was mixed with 1.5 ml of growth media. Following a 24 hr incubation period, the cells were washed and harvested to create a suspension of 2 ⁇ 10 5 cells/ml in serum-free medium.
  • Adhesion assay using P-selectin-coated plates was carried out using a slight modification of a literature protocol. 77
  • the 96-well assay plates were first precoated for 24 hours at 4 °C with 40 ⁇ g/mL of recombinant P-selectin (Sino biological, China) dissolved in 50 ⁇ L of PBS buffer. Separate 6-well plates were seeded at a density of 3 ⁇ 10 5 cells per well and grown overnight at 37 °C. After media removal, cells were then incubated with fresh growth media supplemented with varying proteins concentrations (5 ⁇ M) . In general, 25 ⁇ l of protein stock solution was mixed with 1.5 ml of growth media.
  • the cells were washed and harvested to create a suspension of 5 ⁇ 10 5 cells/ml in serum-free medium.
  • 100 ⁇ l of cell suspension was added to each P-selectin-coated well and then allowed to incubate at 37 °C for 2 hr. The media was then carefully suctioned out from each well, and then washed four times with PBS buffer. Metabolically active adherent cells were then determined using a colorimetric MTS Assay Kit (Abcam) .
  • Millicell Cell Culture Inserts (Merck Millipore) were used to perform the assay, which has a 8.0 ⁇ m pore size and are designed as hanging inserts for 24-well plates. Before use, inserts are coated with matrigel (1 mg/mL) and dried for 2 hr at 37 °C. To conduct the assay, cells were first seeded onto 6-well plates at a density of 3 ⁇ 10 5 cells per well and grown overnight at 37 °C. Following media removal, cells were treated with fresh growth media supplemented with 5 ⁇ M of protein and then incubated for 24 hr at 37 °C. After harvesting, cells were suspended in serum-free media. Approximately 5 ⁇ 10 4 cells were placed in each upper chamber (i.e.
  • Blood agglutination assay The assay was carried out using a slight modification of a literature protocol. 72 Whole-blood was collected from a healthy male volunteer and then centrifuged at 2000 g for 4 minutes. After discarding the supernatant, the cell pellet was washed three times with PBS buffer (pH 7.4) . The pellet of red blood cells was diluted to 2%(v/v) with PBS buffer and then tested immediately. To perform the agglutination assay, varying concentrations of proteins (0-5 nM) were tested by mixing 50 ⁇ l of the cell suspension with 50 ⁇ l of stock protein solutions in V shaped 96-well plates (Sangon) . Following incubation for 1 hr at room temperature, photographs of the wells were taken.
  • Lectin-decorated nanoparticles enhance binding to the inflamed tissue in experimental colitis. J. Control. Release 2014, 188, 9-17 (21) He, X. ; Liu, F. ; Liu, L. ; Duan, T. ; Zhang, H. ; Wang, Z. Lectin-Conjugated Fe2O3@Au Core@Shell Nanoparticles as Dual Mode Contrast Agents for in Vivo Detection of Tumor. Mol. Pharm. 2014, 11 (3) , 738-745 (22) Gao, X. ; Tao, W. ; Lu, W. ; Zhang, Q. ; Zhang, Y. ; Jiang, X. ; Fu, S.
  • Lectin- conjugated PEG–PLA nanoparticles Preparation and brain delivery after intranasal administration. Biomaterials 2006, 27 (18) , 3482-3490 (23) Yin, Y. ; Chen, D. ; Qiao, M. ; Wei, X. ; Hu, H. Lectin-conjugated PLGA nanoparticles loaded with thymopentin: Ex vivo bioadhesion and in vivo biodistribution. J. Control. Release 2007, 123 (1) , 27-38 (24) Sharon, N. ; Lis, H. Lectins: Cell-Agglutinating and Sugar-Specific Proteins.
  • Synthetic prodrug design enables biocatalytic activation in mice to elicit tumor growth suppression.
  • Nat. Commun. 2022, 13 (1) , 39 (37) Vong, K. ; Tahara, T. ; Urano, S. ; Nasibullin, I. ; Tsubokura, K. ; Nakao, Y. ; Kurbangalieva, A. ; Onoe, H. ; Watanabe, Y. ; Tanaka, K. Disrupting tumor onset and growth via selective cell tagging (SeCT) therapy. Sci. Adv. 7 (17) , eabg4038 (38) Tsubokura, K. ; Vong, K. K. H. ; Pradipta, A. R.
  • the Breast 2022, 66, 15-23 (74) Jin, X. ; Demere, Z. ; Nair, K. ; Ali, A. ; Ferraro, G. B. ; Natoli, T. ; Deik, A. ; Petronio, L. ; Tang, A. A. ; Zhu, C. ; et al. A metastasis map of human cancer cell lines. Nature 2020, 588 (7837) , 331-336 (75) Fan, T. -c. ; Lin, W. -d. ; Chang, C. -h. ; Chang, L. -y. ; Chen, Z. -m. ; Khoo, K. -h. ; Yu, A.

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Abstract

L'invention concerne une construction de fusion qui comprend (1) un domaine de ciblage de glycane pouvant se lier de manière spécifique à une molécule de sucre prédéfinie (telle qu'une sur-exprimée sur une certaine surface de cellule cancéreuse), (2) un domaine d'agrégation pouvant s'auto-agréger pour former des multimères parmi des molécules contenant le même domaine, et (3) un domaine de solubilisation éventuel, qui est lié à (1) ou (2) par l'intermédiaire d'un lieur peptidique contenant éventuellement une protéase (telle qu'une sur-exprimée sur un certain site de clivage de surface de cellule cancéreuse). L'invention concerne également l'utilisation de ladite construction dans le traitement du cancer.
PCT/CN2025/109763 2024-08-05 2025-07-22 Thérapie d'agrégation de protéines ciblant le glycane Pending WO2026032010A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080234177A1 (en) * 2005-07-28 2008-09-25 Edwin Bremer Targeting-enhanced activation of galectins
US20170306046A1 (en) * 2014-11-12 2017-10-26 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
CN107406495A (zh) * 2014-05-27 2017-11-28 中央研究院 治疗及检测癌症的组合物及方法
CN110337303A (zh) * 2016-09-20 2019-10-15 圣安德鲁斯大学董事会 细胞调节
WO2023244510A2 (fr) * 2022-06-13 2023-12-21 The Regents Of The University Of California Protéines bi-spécifiques immunothérapeutiques dépendantes du glycane améliorées ayant une demi-vie plus longue

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080234177A1 (en) * 2005-07-28 2008-09-25 Edwin Bremer Targeting-enhanced activation of galectins
CN107406495A (zh) * 2014-05-27 2017-11-28 中央研究院 治疗及检测癌症的组合物及方法
US20170306046A1 (en) * 2014-11-12 2017-10-26 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
CN110337303A (zh) * 2016-09-20 2019-10-15 圣安德鲁斯大学董事会 细胞调节
WO2023244510A2 (fr) * 2022-06-13 2023-12-21 The Regents Of The University Of California Protéines bi-spécifiques immunothérapeutiques dépendantes du glycane améliorées ayant une demi-vie plus longue

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