Classifications

• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • 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
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21062—Subtilisin (3.4.21.62)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

• the present invention is directed to a mutant subtilase cytotoxin B subunit protein (SubB) which can bind glycans having a.2-3 -linked N-glycolylneuraminic acid and glycans having a2-6-linked N-glycolylneuraminic acid.
• the mutant SubB protein has a previously undescribed ability to bind glycans having a.2-6- linked N-glycolylneuraminic acid, while not losing the ability to bind glycans having a.2-3 -linked N-glycolylneuraminic acid.
• This provides a mutant SubB protein that can be used to detect and target a broader spectrum of N-glycolylneuraminic acid- containing glycans than was previously possible.
• An aspect of the invention provides an isolated protein comprising an amino acid sequence of SubB wherein one or more amino acid residues of the amino acid sequence TTSTE (SEQ ID NO:3) are modified, wherein the isolated protein is capable of binding a2-3 -linked N-glycolylneuraminic acid and ot2-6-linked N- glycolylneuraminic acid.
• Another aspect of the invention provides an isolated molecular complex comprising the isolated protein of the first aspect and a glycan comprising a.2-3- linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid.
• composition is a pharmaceutical composition.
• Another aspect of the invention provides a method of isolating a glycan or a cell expressing the glycan, the glycan comprising a.2-3 -linked N-glycolylneuraminic acid and/or an a2-6-linked N-glycolylneuraminic acid, said method including the steps of: combining the isolated protein disclosed herein with a sample to thereby form a complex comprising the isolated protein and a.2-3 -linked N- glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid; and isolating the protein or cell.
• the cell is a tumour cell or a feline blood cell.
• the isolated protein of the first aspect is coupled to a cytotoxic agent.
• Yet another further aspect of the invention provides a host cell comprising the genetic construct of the aforementioned aspect.
• the antibody or antibody fragment binds an epitope comprising one or more modified amino acid residues underlined in the amino acid sequence TTSTE (SEQ ID NO:3).
• kits comprising the isolated protein, isolated nucleic acid, composition, genetic construct and/or antibody, such as for use in detecting a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N- glycolylneuraminic acid or therapeutic targeting of tumour cells expressing a.2-3- linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid, although without limitation thereto.
• the isolated protein or immunogenic fragment has one, two or no more than three amino acid residues in addition to the recited amino acid sequence.
• the additional amino acid residues may occur at the N- and/or C-termini of the recited amino acid sequence, although without limitation thereto.
• indefinite articles “a” and “a «” are not to be read as singular or as otherwise excluding more than one or more than a single subject to which the indefinite article refers.
• a protein includes one protein, one or more proteins or a plurality of proteins.
• FIG. 1 Surface representation of SubB in complex with (A) Neu5Gca2- 3Gaipi-3GlcNAc (determined from a X-ray crystal structure (Byres et al, 2008)) and (B) Neu5Gca2-6Gai i-3Glc (modeled with the X-ray crystal structure). Tri saccharides are shown as a green or cyan stick with red and blue residues representing oxygen and nitrogen, respectively.
• FIG. 1 Surface representation of the wild-type and SubB mutants modeled with Neu5Gca2-6Gai i-3Glc (shown as a cyan stick). The mutated SubB residues are shown as grey sticks and red and blue residues represent oxygen and nitrogen, respectively.
• Figure 4 Lectin overlay assay. Binding of SubBASio6/ATio7 to serial dilutions of human or bovine AGP spotted onto nitrocellulose (total amounts of protein per spot indicated).
• FIG. 7 SubB WT interaction with the Z-Biotech Neu5Ac/Gc glycan array. Neu5Gc glycans are shown in blue, Neu5 Ac are shown in red.
• FIG. 8 SubB AS106/AT107 deletion mutant (Sub2M) interaction with the Z- Biotech Neu5Ac/Gc glycan array. Neu5Gc glycans are shown in blue, Neu5Ac are shown in red.
• Figure 9 Glycans on the Z-biotech array.
• aspects of the invention may relate to purifying, removing or depleting glycans comprising a.2-3 -linked N- glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid, or cells that express these glycans.
• a further aspect of the invention relates to therapeutic uses of the isolated protein for targeted delivery of anti-cancer agents to certain tumour cells.
• isolated material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be partly, substantially or essentially free from, or depleted of, components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. In some embodiments, isolated material may be in enriched, partially purified or purified form.
• a “composition” comprises an isolated protein, nucleic acid, genetic construct, antibody or other molecule together with one or more other components such as water or other solvents, salts, buffering agents and/or stabilizers, although without limitation thereto.
• a “diagnostic” composition may comprise one or more other molecular components that facilitate detection of proteins that comprise a.2-3 -linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid.
• Such components may include enzyme substrates, secondary antibodies, colour reagents, labels and catalysts (e.g "detection reagents”) as will be described in more detail hereinafter.
• glycan is a glycoprotein, glycolipid or other carbohydrate-containing macromolecule, and includes molecules that may be referred to as peptidoglycans, glycoproteins, glycopeptides, glycolipoproteins and the like.
• a particular glycan comprises N-glycolylneuraminic acid (Neu5Gc).
• the glycan may comprise a.2-3 -linked N-glycolylneuraminic acid or a2-6-linked N- glycolylneuraminic acid.
• the a.2-3 -linked N-glycolylneuraminic acid and a2-6-linked N-glycolylneuraminic acids are terminal sialic acids.
• a preferred aspect of the invention provides an isolated protein comprising an amino acid sequence of SubB protein that has one or more modified amino acid residues of the amino acid sequence TTSTE, wherein the isolated protein is capable of binding a glycan comprising a2-3-linked N-glycolylneuraminic acid and a glycan comprising a2-6-linked N- glycolylneuraminic acid.
• a related aspect of the invention provides an isolated molecular complex comprising the isolated protein of the first aspect and a glycan comprising a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N- glycolylneuraminic acid.
• a2-3-linked N-glycolylneuraminic acid and a2-6-linked N-glycolylneuraminic acid means that the isolated protein binds a2-6-linked ⁇ -glycolylneuraminic acid glycans with substantially greater affinity than does a wild-type SubB protein, while also binding a2-3 -linked N- glycolylneuraminic acid glycans with a comparable affinity to that of a wild-type SubB protein.
• the isolated protein binds a2-6-linked N- glycolylneuraminic acid glycans with an affinity of about 5-15 nM, about 7-12 nM or about 8-10 nM. In a particular embodiment, the isolated protein binds a2-3 -linked N-glycolylneuraminic acid glycans with an affinity of about 8-20 nM, 10-18 nM or about 14-16 nM.
• the resultant steric hindrance distorts the docking of the terminal Neu5Gc into the binding pocket, accounting for the significantly poorer binding of a2-6-linked Neu5Gc structures observed on the original glycan array analysis of SubB 8 .
• Modification of one or more residues in the loop enhance binding of a mutant SubB protein to the a.2-6 structure while also allowing binding to a.2-3 structures.
• amino acid deletions or substitutions that reduce or lower the "height" of the loop may be advantageous for improved binding of a2-6-linked Neu5Gc structures.
• "height" may be a function of the distance an amino acid R group projects or extends from the peptide backbone in 3D space (e.g.
• mutant SubB protein comprises the amino acid sequence of SEQ ID NO: 1 :
• the isolated protein comprises a deletion of the amino acid residues underlined in the amino acid sequence TTSTE (SEQ ID NO:3).
• the T107 and E108 deletion mutant can broadly bind glycans including Neu5Ac glycans such as Neu5Ac-a2-6-lac and Neu5Ac-a2- 3-lac (e.g.see Table 1). It is also noted that WT SubB does not detectably bind Neu5Ac-a2-6-lac.
• the T107 and E108 deletion mutant protein may be a useful protein for binding or detecting Neu5Gc glycans such as a2-6-linked N- glycolylneuraminic acid glycans and a.2-3 -linked N-glycolylneuraminic acid glycans and also Neu5Ac glycans such as Neu5Ac-a2-6-lac and Neu5Ac-a2-3-lac.
• Neu5Gc glycans such as a2-6-linked N- glycolylneuraminic acid glycans and a.2-3 -linked N-glycolylneuraminic acid glycans
• Neu5Ac glycans such as Neu5Ac-a2-6-lac and Neu5Ac-a2-3-lac.
• variants, fragments and derivatives of the isolated protein disclosed herein retain an ability to bind glycans terminating in a2-3-linked and a2-6-linked Neu5Gc. In particular embodiments, this is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the ability of the isolated protein of an isolated protein disclosed herein to bind glycans terminating in a2-3-linked and a2-6-linked Neu5Gc.
• the peptide variant disclosed herein may have one or more amino acids deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing biological activity of the peptide (conservative substitutions).
• sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity.
• a “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence.
• the comparison window may comprise additions or deletions ⁇ i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
• sequence identity is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
• a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ⁇ e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison ⁇ i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• sequence identity may be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).
• derivatives proteins or peptides have been altered, for example by conjugation or complexing with other chemical moieties, by post- translational modification ⁇ e.g. phosphorylation, ubiquitination, glycosylation), chemical modification (e.g. cross-linking, acetylation, biotinylation, oxidation or reduction and the like), conjugation with labels (e.g. fluorophores, enzymes, radioactive isotopes) and/or inclusion of additional amino acid sequences as would be understood in the art.
• post- translational modification e.g. phosphorylation, ubiquitination, glycosylation
• chemical modification e.g. cross-linking, acetylation, biotinylation, oxidation or reduction and the like
• conjugation with labels e.g. fluorophores, enzymes, radioactive isotopes
• Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein.
• fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein.
• Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags.
• isolated peptides, variant and/or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis and recombinant DNA technology.
• Chemical synthesis is inclusive of solid phase and solution phase synthesis.
• Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2014), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995- 2014), in particular Chapters 1, 5 and 6.
• An aspect of the invention provides a method of detecting a.2-3 -linked N- glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid, said method including the step of combining the isolated protein disclosed herein with a sample to thereby form a detectable complex comprising the isolated protein of the first aspect and a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N- glycolylneuraminic acid.
• the isolated protein may be used for detecting the presence of a.2-3 -linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid-expressing tumour cells in a patient sample, such as a biopsy, fluid sample, smear or the like.
• the isolated protein may be used for feline blood-typing by detecting blood cells that express N-glycolylneuraminic acid glycans, such as comprising a2-3 -linked N-glycolylneuraminic acid and/or a2-6- linked N-glycolylneuraminic acid, in a feline blood sample.
• glycans comprising a.2-3 -linked N- glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid may be present in a preparation or formulation comprising drugs, antibodies or other therapeutic biomolecules for human administration.
• Recombinant glycosylated drugs, antibodies and other therapeutic biomolecules for human administration are often produced in non-human mammalian cell lines which can synthesize and/or metabolically incorporate the non- human sialic acid N-glycolylneuraminic acid (Neu5Gc).
• Some humans have high levels of circulating anti-Neu5Gc antibodies.
• the clinically effective anti-EGFR mAb Cetuximab may have covalently-bound Neu5Gc.
• Anti- Neu5Gc antibodies from normal humans interact with Cetuximab in a Neu5Gc- specific manner and generate immune complexes in vitro. These antibodies may enhance Cetuximab (or other therapeutic antibody) clearance in vivo.
• inventions provide isolation, depletion or removal of a.2-3 -linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid-containing contaminants from drugs, antibodies or other therapeutic biomolecules for human administration.
• the isolated protein ⁇ e.g. of an isolated protein disclosed herein such as comprising the amino acid sequence of SEQ ID ⁇ : 1), or a fragment or variant thereof, to an agent that facilitates detection of a2-3 -linked N-glycolylneuraminic acid and/or a2- 6-linked N-glycolylneuraminic acid.
• the isolated protein is covalently coupled to a label.
• a labelled secondary binding agent such as an antibody or antibody fragment may be used to detect the isolated protein when bound to glycans comprising a2-3-linked N-glycolylneuraminic acid and/or a.2-6- linked N-glycolylneuraminic acid.
• the enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase or glucose oxidase, although without limitation thereto.
• Appropriate substrates include diaminobanzidine (DAB), permanent red, 3- ethylbenzthiazoline sulfonic acid (ABTS), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 3,3 ',5,5 '-tetramethyl benzidine (T B) and 4- chloro-l-naphthol (4-CN), although without limitation thereto.
• a non-limiting example of a chemiluminescent substrate is LuminolTM, which is oxidized in the presence of HRP and hydrogen peroxide to form an excited state product (3- aminophthalate).
• Radioisotope labels may include 125 I, 1 1 I, 51 Cr and 99 Tc, although without limitation thereto.
• a colloidal metallic or non-metallic particle a dye particle, an organic polymer, a latex particle, a liposome, a minicell or other vesicle containing a signal producing substance and the like.
• the labeled isolated protein may be used in detection systems such as histochemistry, flow cytometry, fluorescence microscopy and ELISAs, body imaging (e.g PET scans) and nuclear medicine although without limitation thereto.
• the invention provides a method of isolating a glycan or a cell expressing the glycan , the glycan comprising a2-3 -linked N- glycolylneuraminic acid and/or an a2-6-linked N-glycolylneuraminic acid, said method including the steps of: combining the isolated protein disclosed herein with a sample to thereby form a complex comprising the isolated protein and a.2-3 -linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid; and isolating the glycan or the cell.
• the term "isolating” preferably refers to purifying, enriching or depleting or removing the glycan comprising a.2-3 -linked N-glycolylneuraminic acid and/or an a2-6-linked N-glycolylneuraminic acid, or cells expressing same.
• the isolated protein (e.g. comprising the amino acid sequence of SEQ ID NO: 1), or a fragment or variant thereof, is coupled to a label as hereinbefore described, which facilitates detection of the glycan comprising a.2-3 - linked N-glycolylneuraminic acid and/or an a2-6-linked N-glycolylneuraminic acid, or cells expressing same.
• a label as hereinbefore described
• a fluorescent label such as hereinbefore described
• the isolated protein disclosed herein, or a fragment or variant thereof may be coupled, bound, affixed or otherwise linked to a substrate that facilitates isolation, enrichment, purification, depletion or removal of a glycan comprising a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N- glycolylneuraminic acid, or cells expressing same.
• the isolated protein disclosed herein, or a fragment or variant thereof, may be coupled, bound, affixed or otherwise linked to a substrate that may be a bead, matrix, cross-linked polymer, gel, particle, surface or other solid or semi-solid substrate.
• a substrate may be or comprise sepharose, agarose, Protein A, Protein G, a magnetic bead, a paramagnetic particle, or sensor chip surface (e.g. for BIACore or surface plasmon resonance).
• tumour cells express glycans comprising a2-3 -linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid, whereas normal cells typically do not express these sugars.
• glycans comprising a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid may be expressed by human carcinomas, with elevated expression detected in breast, ovarian, prostate, colon and lung cancer, although without limitation thereto.
• the isolated protein may be coupled, bound, affixed or otherwise linked to a cytotoxic agent that facilitates binding to, and killing or disabling of, tumour cells that express a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N-glycolylneuraminic acid.
• Non-limiting examples of radionuclides include 211 At, 212 B1, 21 Bi, 125 I, m In, 90 Yt, 19 Pt, 177 Lu, 1 4 Eu and 67 Ga, although without limitation thereto.
• Chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic agents and DNA intercalating agents may include doxorubicin, N-acetyl- ⁇ - calicheamicin, maytansinoids, taxoids, auristatins and duocarmycins, although without limitation thereto. Chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic agents and DNA intercalating agents may be coupled to the isolated protein by a cleavable or non-cleavable linker to form a cleavable conjugate.
• the cleavable conjugate is internalized by the tumour cell where the cleavable linker is cleaved to release the drug into the cell.
• these may be preferred where it is essential that the drug is entirely localized to the targeted tumour cell and there is no "leakage" of the drug from the targeted tumour cell into adjacent cells, tissues or fluids.
• the chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic and DNA intercalating agents may be in the form of a pro-drug which is activated upon internalization inside a targeted tumour cell.
• the isolated protein e.g. comprising the amino acid sequence of SEQ ID NO: l
• the isolated protein may be administered as a pharmaceutical composition.
• the pharmaceutical composition comprises a pharmaceutically- acceptable carrier, diluent or excipient.
• pharmaceutically-acceptable carrier diluent or excipienf is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.
• a variety of carriers well known in the art may be used.
• These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
• a useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.
• any safe route of administration may be employed for providing a patient with the composition of the invention.
• oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.
• Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines.
• Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
• compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
• Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
• the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
• compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective.
• the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
• the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
• Another aspect of the invention provides an antibody or antibody fragment that binds the isolated protein disclosed herein.
• the antibody or antibody fragment does not bind a wild-type SubB protein (such as comprising the amino acid sequence of SEQ ID NO: 2), or binds with at least 5 or 10-fold lower affinity compared to the affinity with which it binds the isolated protein disclosed herein (such as comprising the amino acid sequence of SEQ ID NO: l).
• the antibody or antibody fragment binds an epitope of SEQ ID NO: l comprising the one or more modified amino acid residues of the amino acid sequence TTSTE.
• Antibody fragments include Fab and Fab'2 fragments, diabodies and single chain antibody fragments ⁇ e.g. scVs), although without limitation thereto.
• an antibody comprises respective light chain and heavy chain variable regions that each comprise CDR 1, 2 and 3 amino acid sequences
• the antibody or antibody fragment may comprise at least a portion of a CDR1, 2 and/or 3 amino acid sequence
• a preferred antibody fragment comprises at least one entire light chain variable region CDR and/or at least one entire heavy chain variable region CDR.
• Antibodies and antibody fragments may be polyclonal or preferably monoclonal.
• Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example described in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN FMMUNOLOGY, by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated the isolated protein (e.g. comprising the amino acid sequence of SEQ ID NO: l) or a fragment or variant thereof.
• antibodies may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell.
• Recombinant synthetic antibody or antibody fragment heavy and light chains may be co-expressed from different expression vectors in the same host cell or expressed as a single chain antibody in a host cell.
• Non-limiting examples of recombinant antibody expression and selection techniques are provided in Chapter 17 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY supra and Zuberbuhler et al., 2009, Protein Engineering, Design & Selection 22 169.
• Antibodies and antibody fragments may be modified so as to be administrable to one species having being produced in, or originating from, another species without eliciting a deleterious immune response to the "foreign" antibody. In the context of humans, this is “humanization” of the antibody produced in, or originating from, another species.
• Such methods are well known in the art and generally involve recombinant "grafting" of non-human antibody complementarity determining regions (CDRs) onto a human antibody scaffold or backbone.
• CDRs complementarity determining regions
• the antibody or antibody fragment is labeled. Labels may be as hereinbefore described.
• the invention also provides an isolated nucleic acid encoding the isolated protein disclosed herein, or a fragment or variant thereof.
• the isolated nucleic acid encodes the isolated protein comprising the amino acid sequence set forth in SEQ ID NO: l, or a fragment or variant thereof.
• nucleic acid designates single- or double-stranded DNA and RNA.
• DNA includes genomic DNA and cDNA.
• RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA.
• Nucleic acids may also be DNA-RNA hybrids.
• a nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.
• a nucleic acid variant may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to an nucleotide sequence encoding SEQ ID NO: l.
• a nucleic acid variant may hybridize to a nucleotide sequence encoding SEQ ID NO: l under high stringency conditions.
• T m of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases.
• the isolated nucleic acid is in a genetic construct that comprises the isolated nucleic acid operably linked or connected to one or more other genetic components.
• a genetic construct may be suitable for therapeutic delivery of the isolated nucleic acid or for recombinant protein production in a host cell.
• the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and/or expression of the nucleic acid or an encoded protein of the invention.
• the genetic construct is an expression construct.
• the expression construct comprises the nucleic acid of the invention operably linked to one or more additional sequences in an expression vector.
• An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.
• operably linked is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription.
• said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, polyadenylatioin sequences, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.
• the expression construct may also include an additional nucleotide sequence encoding a selection marker such as amp R , neo R or kan R , although without limitation thereto.
• the expression construct may be in the form of plasmid DNA, suitably comprising a promoter operable in an animal cell ⁇ e.g. a CMV, an a A- crystallin or SV40 promoter).
• the nucleic acid may be in the form of a viral construct such as an adenoviral, vaccinia, lentiviral or adeno- associated viral vector.
• the invention provides a host cell transformed with a nucleic acid molecule or a genetic construct described herein.
• Suitable host cells for expression may be prokaryotic or eukaryotic.
• suitable host cells may include but are not limited to mammalian cells ⁇ e.g. HeLa, Cos, NIH-3T3, HEK293T, Jurkat cells), yeast cells ⁇ e.g. Saccharomyces cerevisiae), insect cells (e.g. S/9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli.
• mammalian cells ⁇ e.g. HeLa, Cos, NIH-3T3, HEK293T, Jurkat cells
• yeast cells ⁇ e.g. Saccharomyces cerevisiae
• insect cells e.g. S/9, Trichoplusia ni
• plant cells e.g. Chlamydomon
• kits comprising the isolated protein, isolated nucleic acid, genetic construct and/or antibody, such as for expression of the isolated protein, use in detecting a2-3-linked N-glycolylneuraminic acid and/or a.2- 6-linked N-glycolylneuraminic acid or therepeutic targeting of tumour cells expressing a2-3-linked N-glycolylneuraminic acid and/or a2-6-linked N- glycolylneuraminic acid, although without limitation thereto.
• kits for expression of the isolated protein may comprise a genetic construct encoding the isolated protein, suitable host cells for transfection and expression of the isolated protein and instructions for use.
• Wild-type SubB was found to have high affinity for a.2-3 -linked Neu5Gc-lactose and free Neu5Gc, as predicted from the glycan array result, with nanomolar binding affinities observed. No binding was observed for the a2-6-linked Neu5Gc-lactose (tested to a maximum concentration of 25 ⁇ ) and 2.2 ⁇ affinity was observed for a2-3-linked Neu5Ac - a more than 300-fold decrease in binding compared to the equivalent Neu5Gc structure. The wild-type SubB also had a 13 -fold reduced binding affinity for human AGP compared to bovine AGP.
• SubBAsio6/ATio7 exhibited the optimum combination of enhanced Neu5Gc vs Neu5Ac discrimination and the capacity to recognise both a2-3- and a2-6-linked Neu5Gc structures.
• the SubBATio7/AEio8 deletion mutant bound a2-6-linked N-glycolylneuraminic acid glycans with substantially greater affinity than wild-type SubB protein, while also binding a2-3 -linked N-glycolylneuraminic acid glycans.
• SubBATio7/AEio8 can broadly bind Neu5Ac glycans such as Neu5Ac- a2-6-lac, which are not detectably bound by either wild-type SubB or
• the anti-Neu5Gc antibody produced in chicken was used as a control and showed less selectivity and lower affinity for Neu5Gc containing glycans than any of the SubB proteins tested.
• FIG. 9 The various Neu5 Ac and Neu5Gc glycan structures analysed in the Z-biotech glycan arrays are shown in FIG. 9 and an example of an array in FIG. 6.
• Table 3 provides the code linking the glycans of FIG. 9 with the array data in FIGS 7 and 8.
• the array data summarized in FIG. 7 show that binding to Neu5Gc structures is preferred by the wild-type SubB but there are 4/40 Neu5Ac glycans that are bound with greater than 5000 fluorescence units above background and 14/41 Neu5Gc structures that have binding below 5000. All Neu5Ac structures register some binding above background. As also evident in FIG. 8, binding to Neu5Gc structures is preferred by SubB 2M.
• SubBAsio6/ATio7 mutant was significantly improved for the recognition Neu5Gc containing structures compared to the wild-type SubB.
• SubBAsio6/ATio7 also had no difference in its ability to bind a2-3-linked Neu5Gc or a2-6-linked Neu5Gc structures, making it a significant improvement over the wild-type protein.
• Further modifications of the SubB protein outside of the SI 06 and T107 amino acids produced no significant improvement in specificity.
• the SubBAsio6/ATio7/Eio8D mutant protein which is the SubBAsio6/ATio7 protein with a E108D mutation also added, was less able to distinguish a2-3-linked Neu5Gc from a2-3-linked Neu5Ac than SubBAsio6/ATio7 and had stronger binding to the human al-Acid glycoprotein than the SubBAsio6/ATio7 mutant (24 fold more protein bound by SubBAsio6/ATio7/Eio8D than SubBAsio6/ATio7)-
• the SubBATio7/AEios deletion mutant not only bound a.2- 6-linked N-glycolylneuraminic acid glycans and a.2-3 -linked N-glycolylneuraminic acid glycans but also Neu5Ac glycans such as Neu5Ac-a2-6-lac and Neu5Ac-a2-3- lac, which are
• the three-dimensional structure of the SubB mutants were modeled using Phyre2 24 .
• Neu5GCa2-6Galpl-3Glc was acquired from PDB ID: 4EN8 25 and modeled into the SubB and SubB mutant structures manually using Coot 26 .
• Mutations were introduced into the subB coding sequence (close to the 3 ' end) by direct high-fidelity PCR using the forward primer pETSubBF and the respective mutant-specific reverse primers listed in Table 2.
• PCR products were cloned into t e BamHI and Xhol sites of pET-23(+) (Novagen) and transformed into E. coli BL21(DE3).
• SubB derivatives were expressed and purified as His6 -tagged fusion proteins by Ni-NTA affinity chromatography, as previously described 4 . Proteins were >95% pure as judged by SDS-PAGE and Coomassie blue staining.
• AGP from human plasma (Sigma-Aldrich G9885) and bovine plasma (Sigma-Aldrich G3643) (lmg in 6M guanidinium chloride, 50 mM Tris-HCl pH8) was reduced and alkylated with 10 mM dithiothreitol and 25 mM acrylamide, respectively. Protein was then precipitated by adding 4 volumes of 1 : 1 methanol: acetone, incubating in -20°C for 16 h and then centrifuged (18,000 rcf, 10 min) to collect the pellet.
• the precipitated protein was resuspended in 50 ⁇ _, of 50 mM Tris-HCl pH8 and digested (37°C, 16 h) with 1 ⁇ g trypsin (Trypsin Gold, Promega). Digested peptides were then desalted with CI 8 ZipTips (Millipore).
• FITC-labelled normal human serum or bovine serum was added to wells coated with SubB or SubBAsio6/ATio7 and wells were incubated for 1 hour at room temperature. Wells were washed 3 times with PBS-T. ⁇ of PBS was added to each well before the fluorescence was measured at 485/535 nm. Fluorescence unit values are shown as the mean of duplicates +/- SD, with the mean fluorescence units obtained for wells containing all reagents except for the SubB proteins subtracted. Any negative value was considered as 0.
• glycan array slides were printed on SuperEpoxy 3 (Arrayit) activated substrates using an Arrayit Spotbot Extreme contact printer as previously described 28 .
• SubB proteins were pre-complexed with anti-His tag antibody (Cell signalling) and Alexa555 secondary and tertiary antibodies (rabbit anti-mouse; goat anti-rabbit) at a ratio of 2: 1 :0.5:0.25 in a final volume of 500 [iL.
• This 500 [iL antibody protein complex was added to a 65 ⁇ _, gene frame (Thermo Scientific) without a coverslip. Washing and analysis was performed as previously described 27 .
• Neu5Ac/Neu5Gc Glycan arrays were obtained from Z-biotech (http://www.zbiotech.com/neu5gc-xenoantigen- microarray.html). Arrays were preformed as per manufacturer's instructions with a total of 2 ⁇ g of protein applied to each of the subarray areas. Detection was with mouse anti-His IgG (1 : 1 molar ratio with protein), rabbit anti-mouse Alexa 555 IgG (0.5 molar amount of mouse IgG) and goat anti-rabbit Alexa 555 IgG (0.5 molar amount of rabbit IgG). Proteins were incubated for 1 hour and washed 3 times in lxPBS. Slides were scanned on an Innoscan l lOOAL using 488, 532 and 647 lasers. Arrays were analysed with Mapix software. All data was taken from the 532 laser channel and background subtracted fluorescence was used in the analysis.
• Kidney Int Lofling, J.C, Paton, A.W., Varki, N.M., Paton, J.C. & Varki, A.
• a dietary non-human sialic acid may facilitate hemolytic-uremic syndrome. Kidney Int

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