WO2023132795A2

WO2023132795A2 - Modified bacterial glycans and conjugates thereof

Info

Publication number: WO2023132795A2
Application number: PCT/SG2023/050010
Authority: WO
Inventors: Lok To SHAM; Tong Su
Original assignee: National University of Singapore
Current assignee: National University of Singapore
Priority date: 2022-01-05
Filing date: 2023-01-05
Publication date: 2023-07-13
Anticipated expiration: 2024-07-05
Also published as: WO2023132795A3

Abstract

The invention relates generally to glycobiology. In particular, the specification teaches a modified bacterial glycan comprising a heterologous antigen.

Description

Modified Bacterial Glycans and Conjugates thereof

Field of Invention

Background

Paragloboside is the precursor of many biologically important glycans, such as ABO blood group antigens, Lewis antigens, and the Galili (a-Gal) antigen. It is technically challenging to produce a sufficient amount of these antigens for medical use. For example, anti-A and anti-B antibodies can cause hyperacute or acute rejection when the patient’s blood group is incompatible with the donor (i.e., ABOi). These antibodies led to complement deposition on the surface of the graft, resulting in severe inflammation and apoptosis. The ABOi-mediated rejection can be alleviated by removing the anti- A/B antibodies using the Glycosorb- ABO column prior to the surgery, which costs -US$19, 000 per piece. Reusing the column is possible, but it generates concerns of blood-borne infections, an increased rate of graft rejection, and ethical issues. The alternatives, such as apheresis and splenectomy, are laborious and associated with a long-term increased risk of infections.

The a-Gal epitope (Gal(al,3)Gal(pi,4)GlcNAc) is a major obstacle to xenotransplantation, especially for porcine tissues. Other than the Old World monkeys, apes, and humans, all mammalian nucleated cells contain roughly 1 to 30 million copies of a-Gal epitope on the surface. This epitope is recognized by one of the most abundant natural antibodies in humans known as the anti-Gal antibodies. As much as 1% of all B- cells are producing anti-Gal antibodies, which can constitute up to 4% of total immunoglobulins. When bound to the graft, anti-Gal antibodies will ultimately trigger hyperacute rejection. As the graft continuously induces anti-Gal IgG production by -100-fold in two weeks, there is no current technology that can stop anti-Gal production. Various possibilities, such as destroying the a-Gal epitope by enzymatic digestion, knocking out the ggta-1 and other al, 3 glycosyltransferases, and removing live cells from the implant tissues, have not succeeded clinically. The modified enzyme often could not penetrate deep enough into the tissue to completely remove the a-Gal epitope. Deletion of the genes responsible to produce the a-Gal epitope is challenging due to gene redundancy. Finally, striping live cells will tremendously reduce the utility of the transplant.

The capsular polysaccharide (CPS) of Streptococcus pneumoniae is a surface glycan that protects the bacterial cell from host immunity. The structure of CPS is extremely diverse, with at least 100 serotypes identified to date. Among them is the serotype 14 CPS, in which the repeating unit is strikingly similar to paragloboside (nLC4). The serotype 14 repeating units are linked with a pi,6 linkage at the third N- acetylglucosamine residue to form a polymer, thereby exposing the rest of the carbon positions and a terminal P-linked galactose residue for glycoengineering.

It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

Summary

Disclosed herein is a bacterial glycan modified to comprise at least one heterologous antigen.

Disclosed herein is a method of producing a modified glycan comprising at least one heterologous antigen, the method comprising a step of reacting a bacterial glycan with at least one glycosyltransferase and at least one donor molecule.

Disclosed herein is a modified glycan obtained according to a method as defined herein.

Disclosed herein is a conjugate comprising a modified glycan as defined herein and a carrier protein. Disclosed herein is a composition comprising a modified glycan or a conjugate as defined herein.

Disclosed herein is a method of reducing or eliminating one or more components from a blood product from a subject, the method comprising a) contacting the blood product with a modified glycan as defined herein, wherein the modified glycan is immobilized on or within a substrate, so as to remove the one or more components from the blood product.

Disclosed herein is a kit for reducing or eliminating one or more components from a blood product from a subject, the kit comprising a substrate, wherein the substrate comprises a modified glycan as defined herein immobilized on or within the substrate.

Disclosed herein is a method for treating a subject of a disease or condition, the method comprising administering a modified glycan or conjugate as defined herein to the subject.

Brief Description of Drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1. A schematic of synthesizing blood group antigens and the a-Gal epitope from S. pneumoniae CPS. The glycans found in humans were highlighted by the blue shade box, the a-Gal epitope is boxed in orange, and the Lewis antigens are boxed in purple.

Figure 2. Purification and characterization of Ggta-1. (A) Strain LEMO21/pCS190 [PT7 -.-.his-ggta-l was grown in LB medium, induced, and lysed. The cell lysate was centrifuged, and the supernatant (input) was loaded on a TALON column. Once the protein was bound, the lysate was removed (FT) and the column was washed twice (W 1 and W2) before elution. (B) The enzymatic activity was determined by measuring the release of UDP from UDP-Gal. (C) The rate of UDP released is proportional to the amount of Ggta- 1 added to the GT reaction. Figure 3. Modifying serotype 14 CPS with Ggta-1 generates the a-Gal epitope. Strain NUS0661 (CPS14) was grown in BHI broth. Cells were collected by centrifugation, boiled in SDS, and treated with Ggta-1. The untreated control (A) and the Ggta-1 treated sample (B) were stained with the indicated lectin conjugates and visualized with fluorescent microscopy. The samples were diluted and spotted on a nitrocellulose membrane together with the a-gal control. The membrane was then blotted with ECL (C) or IB4 (D) lectins to visualize a or P galactoside, respectively.

Figure 4. Isolation of CPS. (A) ID NMR of the purified CPS from the new protocol (blue) and the commercial standard (red). The polysaccharide isolated was analyzed by 2D NMR (HSQC) experiments.

Figure 5. Direct labeling of purified CPS with Ggta-1. Purified CPS was treated with Ggta-1 as described in the legend of Fig. 3. The blot was stained with IB4 (A) or ECL (B), respectively. An overexposed blot was provided on the right for the visualization of weaker signals.

Figure 6. The a-gal epitope synthesized is not cytotoxic. Heat -killed CPS was treated with GgtA-1 as described in the legend of Fig. 3. A549 cells were incubated with WT control (CPS only) and the modified glycan (Ggta-1 treated), and cell viability was evaluated at 5hrs, 24hrs, and 48hrs post-incubation.

Detailed Description

The present specification teaches a bacterial glycan modified to comprise at least one heterologous antigen.

The terms “antigen” and “epitope” are well understood in the art and refer to the portion of a macromolecule which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell antigen receptor. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa, and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. The antigen may be a fragment or a mimic of a naturally-occurring antigen.

The term “heterologous antigen” as used herein may refer to an antigen that is foreign to a bacterium from which a glycan is isolated or obtained. For example, one may modify a bacterial glycan to comprise at least one mammalian antigen.

“Modified bacterial glycan” refers to a bacterial glycan that is modified so as to have a structure that is distinct from a native form of the bacterial glycan (i.e., the natural form of the glycan found on the bacteria).

The heterologous antigen as defined herein may comprise a mammalian antigen, such as a human or porcine antigen. The mammalian antigen may comprise an ABO blood group antigen, a Lewis antigen, or an a-Gal antigen.

The mammalian antigen may comprise an ABO blood group antigen. The ABO blood group antigen may comprise a Fuc(al,2)Gal motif linked to GlcNAc. The ABO blood group antigen may be selected from the group consisting of a blood group antigen A, a blood group antigen B, and a blood group antigen H.

The bacterial glycan may comprise paragloboside mimic (i.e. a glycan that is structurally similar to paragloboside (nLC4). The paragloboside mimic may be a bacterial paragloboside mimic. The bacterial glycan may comprise Gal(pi,4)GlcNAc(pi,3)Gal(pi,4)Glc. The bacterial glycan may comprise a Streptococcus pneumoniae serotype 14 CPS. The bacterial glycan may comprise a repeating unit of the following structure:

In one embodiment, the bacterial glycan is an isolated or purified bacterial glycan. The bacterial glycan may be attached to other components in the bacterial cell (e.g. a protein or lipid). The bacterial glycan may be pre-conjugated to a protein before it is modified.

The term “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical, synthetic or recombinant form.

“Purified” refers to glycans substantially separated from the various protein and lipid components naturally associated with the glycan. Residual foreign components in the purified oligosaccharide do not interfere with the use of the purified material as an antigen. The term “purified” does not exclude synthetic oligosaccharide preparations retaining artifacts of their synthesis; nor does it exclude preparations that include some impurities, so long as the preparation exhibits reproducible data, for example, molecular weight, sugar residue content, sugar linkages, chromatographic response, and immunogenic behavior.

The specification also teaches a method of producing a modified bacterial glycan comprising at least one heterologous antigen.

Provided herein is a method of producing a modified bacterial glycan comprising at least one heterologous antigen, the method comprising the step of contacting a bacterial glycan with at least one glycosyltransferase and at least one donor molecule.

The method may comprise a step of isolating the bacterial glycan from bacteria. The bacterial glycan may be further purified. The bacterial glycan may be attached to a protein (i.e. a glycoprotein) or lipid. Alternatively, the bacterial glycan may be preconjugated to a protein or lipid. The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

The bacterial glycan may be modified by addition of at least one donor molecule to the glycan, through the action of at least one glycosyltransferase, to produce a mammalian antigen in the modified glycan.

"Glycosyltransferase" or "glycosyltransferase polypeptide" refers to a polypeptide having an enzymatic capability of transferring a donor molecule to an acceptor molecule

The donor molecule may be a monosaccharide derivative. The monosaccharide derivative may be a nucleotide-activated monosaccharide. The monosaccharide may be selected from the group consisting of galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), N- acetylneuraminic acid (Sia) and glucose (Glu).

The glycosyltransferase polypeptide may be selected from the group consisting of alpha- 1,3 galactosyltransferase (Ggtal), alpha-1,2 fucosyltransferase (FutC), alpha-1,3 fucosyltransferase (FutT), alpha-2,3 sialyltransferase (CpsK) and alpha- 1,3 N- acetylgalactosamine transferase (HMU_12050), or a functional fragment thereof.

The Ggtal glycosyltransferase polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. The FutC glycosyltransferase polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. Alternatively, the FutC glycosyltransferase may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6, 7 or 8. The HMU-12050 glycosyltransferase polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3. The FutT glycosyltransferase polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4. The CpsK glycosyltransferase polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5.

The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, 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, and I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) 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.

The term "at least 70% sequence identity" may refer to a sequence identity of at least 75%, 80%, 85%, 90%, 95%, 99%, 100% or anywhere in-between.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with Ggtal and UDP-Gal.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with Ggtal and UDP-Gal. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutC and GDP-Fuc. In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutC and GDP-Fuc.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutC and GDP-Fuc. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with HMU_12050 and GDP-GalNAc.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutT and GDP-Fuc.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutT and GDP-Fuc. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS with CpsK and CMP- NcuNAc.

In one embodiment, the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutT and GDP-Fuc. The method may comprise contacting the Streptococcus pneumoniae serotype 14 CPS as defined herein with FutC and GDP-Fuc.

The method as defined herein may comprise a prior step of isolating the glycan from bacteria.

The method as defined herein may further comprise conjugating the modified glycan to a protein, such as a carrier protein. The method as defined herein may further comprise immobilizing the modified glycan onto a surface. The modified glycans may be immobilized onto the surface via non- covalent interactions or covalent interactions (e.g. via chemical linkers).

The modified glycans may be provided on a solid support, such as a membrane, resin, solid carrier, or other solid phase material. A solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or metal, such as gold or platinum. The configuration of a solid support can be in the form of beads, spheres, particles, granules, a gel, a membrane, or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other containers, vessel, feature, or location.

Provided herein is a modified glycan obtained according to a method as defined herein.

The bacterial glycan as defined herein may be conjugated to a protein or a lipid. The bacterial glycan may be present on a protein (i.e. a glycoprotein). The bacterial glycan may be present on the surface of a cell.

The term “conjugate” as used herein refers to a covalent or non-covalent association of bacterial glycan as defined herein and another molecule such as a carrier protein, regardless of the method of association.

To enhance immunogenicity, a modified glycan as defined herein may be conjugated to a carrier. Conjugation to carrier proteins is well known in the art. For example, the modified glycan may be conjugated to bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. Other suitable carrier proteins include the bacterial outer membrane proteins, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors etc. Provided herein is a protein comprising a bacterial glycan as defined herein. Provided herein is also a cell comprising a bacterial glycan as defined herein.

Provided herein are also compositions for in vitro modification of a bacterial glycan. The compositions may include a glycosyltransferase polypeptide and a physiologically acceptable solution free of divalent metal co-factors. The glycosyltransferase polypeptide may be selected from the group consisting of alpha- 1,3 galactosyltransferase (Ggtal), alpha-1,2 fucosyltransferase (FutC), alpha-1,3 fucosyltransferase (FutT), alpha-2,3 sialyltransferase (CpsK) and alpha- 1,3 N- acetylgalactosamine transferase (HMU_12050), or a functional fragment thereof.

Provided herein is a composition comprising Ggta-1. Provided herein is a composition comprising FutC. Provided herein is a composition comprising HMU_12050. Provided herein is a composition comprising FutT. Provided herein is a composition comprising Ggta-1 and FutC. Provided herein is a composition comprising FutC and HMU_12050. Provided herein is a composition comprising FutT and CpsK (GBS). Provided herein is a composition comprising FutT and FutC.

Provided herein is a method of reducing or eliminating one or more components from a blood product from a subject, the method comprising a) contacting the blood product with a modified glycan as defined herein, wherein the modified glycan is immobilized on or within a substrate, so as to remove the one or more components from the blood product.

The blood product may be whole blood, blood plasma, or completely or partially purified blood plasma. The blood product may be returned to the subject or collected for further use following step a).

Whole blood is simply the fluid that circulates throughout an organism, such as a human. Whole blood includes components including cells (e.g., red blood cells, white blood cells), proteins (e.g., insulin, immunoglobulins, albumin), fatty acids, and carbohydrates (e.g., cholesterol and glucose) suspended in a liquid called plasma. Blood can be treated with an anticoagulant. Thus, the term "blood product" includes, without limitation, whole blood treated with sodium citrate (an anticoagulant), platelet-rich plasma treated with citric acid, and packed red blood cells treated with heparin.

Whole blood can be processed to separate and collect individual components from the blood. For example, red and white blood cells can be removed, leaving the non-cellular components suspended in plasma. Apheresis systems, such as plateletpheresis systems or plasmapheresis systems are commercially available and used to extract desired components (e.g., platelets or plasma) from whole blood from a donor. In some embodiments, the component-reduced blood is returned to the donor by the apheresis system.

Since all the blood-borne molecules (e.g., proteins, lipids, carbohydrates, etc.) not contained within cells and/or attached to the surface of cells are suspended in plasma, the plasma portion of blood will contain these molecules. The proteins in the plasma, which are referred to as blood plasma proteins or simply plasma proteins, are varied and have multiple roles. For example, all of the factors of the coagulation cascade (e.g., fibrinogen, plasminogen thrombin, Factor XIII, Factor IX, Factor VIII, and Factor V) are plasma proteins. Other plasma proteins include, without limitation, the complement proteins (e.g., complement component 3 and complement component 4), albumin, globulins (e.g., alpha 1 globulins and gamma globulins), C-reactive protein (CRP), lipoproteins (e.g., HDL, LDL), and various hormones and enzymes. Albumins account for 55% of blood plasma proteins, and fibrinogen comprises another 7%.

Provided herein is a kit for reducing or eliminating one or more components from a blood product from a subject, the kit comprising a substrate, wherein the substrate comprises a modified glycan as defined herein immobilized on or within the substrate.

The substrate may, for example, comprise a blood group antigen A, a blood group antigen B, or a blood group antigen H for removing a corresponding antibody from the blood product. The substrate may, for example, comprise a Lewis antigen, or an a-Gal antigen, for removing a corresponding antibody from the blood product that recognizes the Lewis antigen, or the a-Gal antigen. Provided herein is a composition comprising a modified bacterial glycan as defined herein or a conjugate as defined herein. Representative compositions may include a buffer, which is selected according to the desired use of the modified bacterial glycan, and may also include other substances appropriate to the intended use. Where the intended use is to elicit or increase immune response, the composition is referred to as an “immunogenic” or “immunomodulating” composition. Such compositions include preventative compositions (i.e., compositions administered for the purpose of preventing a condition) and therapeutic compositions (i.e., compositions administered for the purpose of treating conditions). An immunomodulating composition of the present invention may therefore be administered to a recipient for prophylactic, ameliorative, palliative, or therapeutic purposes.

As used herein, the term “immunogenic” when used in the context of a given agent such as, for example, a modified glycan, a glycoprotein, an antigen, or an epitope, means that the agent has the capability to induce an immune response, enhance an existing immune response, or alter an existing immune response, either alone, or acting in combination with other agent(s). The immune response may include a humoral and/or cellular immune response in a subject. It will be understood that “inducing” an immune response as contemplated herein includes stimulating an immune response and/or enhancing a previously existing immune response.

Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for intended use. In some instances, the composition can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc. Pharmaceutical compositions of the present invention may be in a form suitable for administration by injection, in a formulation suitable for oral ingestion (such as, for example, capsules, tablets, caplets, elixirs), in the form of an ointment, cream, or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, or in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

Supplementary active ingredients such as adjuvants or biological response modifiers can also be incorporated into pharmaceutical compositions of the present invention. Although adjuvant(s) may be included in pharmaceutical compositions of the present invention they need not necessarily comprise an adjuvant. In such cases, reactogenicity problems arising from the use of adjuvants may be avoided.

In general, adjuvant activity in the context of a pharmaceutical composition of the present invention includes, but is not limited to, an ability to enhance the immune response (quantitatively or qualitatively) induced by immunogenic components in the composition. This may reduce the dose or level of the immunogenic components required to produce an immune response and/or reduce the number or the frequency of immunizations required to produce the desired immune response.

Any suitable adjuvant may be included in a pharmaceutical composition of the present invention. For example, an aluminum-based adjuvant may be utilized. Suitable aluminum-based adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, and combinations thereof. Other specific examples of aluminium- based adjuvants that may be utilized are described in European Patent No. 1216053 and United States Patent No. 6,372,223. Other suitable adjuvants include Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminium salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; oil in water emulsions including those described in European Patent No. 0399843, United States Patent No. 7,029,678 and PCT Publication No. WO 2007/006939; and/or additional cytokines, such as GM-CSF or interleukin-2, -7, or -12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF) monophosphoryl lipid A (MPE), cholera toxin (CT) or its constituent subunit, heat-labile enterotoxin (LT) or its constituent subunit, toll-like receptor ligand adjuvants such as lipopolysaccharide (LPS) and derivatives thereof (e.g., monophosphoryl lipid A and 3-Deacylated monophosphoryl lipid A), muramyl dipeptide (MDP) and F protein of Respiratory Syncytial Virus (RSV).

Provided herein is a composition comprising a modified bacterial glycan as defined herein or a conjugate as defined herein for use as a medicament.

Provided herein is a method for treating a subject of a disease or condition, the method comprising administering a modified glycan or a conjugate as defined herein to the subject.

The modified glycan or conjugate as defined herein may be administered to the subject in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of a disease or a condition.

The disease or condition may comprise a cellular, tissue, or organ transplant rejection or a blood transfusion rejection.

The term “treating" as used herein may refer to (1) delaying the appearance of one or more symptoms of the condition; (2) inhibiting the development of the condition or one or more symptoms of the condition; (3) relieving the condition, i.e., causing regression of the condition or at least one or more symptoms of the condition; and/or (4) causing a decrease in the severity of the condition or of one or more symptoms of the condition. By “subject” or “patient” it meant any single subject for which therapy is desired, including humans, cattle, horses, pigs, goats, sheep, dogs, cats, guinea pigs, rabbits, chickens, insects, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease or subjects involved in epidemiological studies, or subjects used as controls.

As used herein, the term "effective amount" relates to an amount of glycan or conjugate which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months, or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health, and weight of the patient to be treated.

The glycan or conjugate of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well-known to those skilled in the art. The composition may contain any suitable carriers, diluents, or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents, and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). The composition may be provided in a substance that protects it from the action of acids and other natural compounds that may inactivate it.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

RESULTS AND DISCUSSIONS

Purification and biochemical characterization of GTs

The synthesis scheme is outlined in Fig. 1. The invention provides methods for modifying the serotype 14 CPS (CPS14) of S. pneumoniae with various glycosyltransferases (GTs) either individually or in combination, to produce the Galili antigen, Lewis antigens, and blood group ABO antigens. These enzymes comprise Ggta-1, FutC, FutT, CpsK, and HMU_12050. Ggta-1 installs an al, 3 linked Gal to the terminal Gal residue of CPS 14, thereby generating the Galili antigen. When combined with FutC, which adds an al, 2 linked fucose residue on the same Gal residue, the blood group B antigen is formed. Similarly, when the Ggta-1 treated CPS 14 is modified by HMU_12050 in Helicobacter mustelae that links an al, 3 GalNAc to the same site, it generates the blood group A antigen. FutT in H. pylori installs an al, 3 linked fucose to the third GlcNAc residue of CPS 14, thereby generating the Lewis X antigen. FutT when combined with FutC, which installs an al, 2 linked fucose residue on the terminal Gal residue will generate the Lewis Y antigen. When FutT is combined with CpsK (GBS), which installs an a2,3 linked N-acetyl neuraminic acid (NeuNAc or sialic acid) residue on the terminal Gal residue will generate the sialyl Lewis X antigen. The open reading frames of ggta-1 from Bos taurus and hmu_12050 from H. mustelae were codon optimized with their DNA sequence synthesized and cloned, where the N-termini of the GTs are fused with a hise tag. A similar strategy was used to clone FutC and FutT, except these were done by PCR using genomic DNA of H. pylori as the DNA template, followed by restriction digestion with BamHI and Xhol. The recombinant plasmids were validated by PCR and sequencing and introduced into strain LEMO21(1DE3) or Rosetta2 for expression. Ggta-1 was affinity purified and the yield was -3 mg per liter of culture. The purity of Ggta-1 was -95%, as judged by the intensity of contaminating protein bands on a Coomassie blue stained SDS-PAGE gel (Fig. 2).

To test whether the purified Ggta-1 is active, cells of strain NUS0661 [D39 rpsLl cpsl4] were boiled in SDS and washed extensively to remove cellular components. The total cell wall extracted was incubated with Ggta-1 and UDP-Gal, the latter served as a glycan donor. The released UDP was quantitated using a luminescence assay to measure the reaction kinetics. The specific activity of Ggta-1 is -381 units per mg of protein, where one unit is defined as one pmole galactose transferred to an acceptor molecule per minute at 37°C (Fig. 2), assuming the number of galactose molecules transferred equals the number of UDP released. The Ggta-1 preparation has a significantly higher catalytic activity than reported previously, which is -10 units per mg of protein.

To demonstrate the resulting glycan is indeed the Galili antigen, the modified CPS14 was stained with fluorescently labeled lectins ECL and GSL. ECL binds to a terminal pi,4 linked Gal and therefore labels CPS14 robustly. In contrast, GSL interacts with glycans with a terminal al, 3 Gal and can be used to detect the a-Gal epitope. Indeed, fluorescent microscopy validated that the Ggta-1 treated CPS 14 contains the a-Gal antigen (Fig. 3). This was confirmed by dot-blotting using BSA conjugated with a-Gal epitope as a control (Fig. 3). A profuse amount of a-Gal epitope (-4.1 g per liter of culture) could be synthesized with this method.

Purification of serotype 14 CPS

Removing other contaminants such as peptidoglycan and LPxTG cell wall proteins may be necessary for therapeutics. The traditional approach to purifying CPS requires a large volume of organic solvent to precipitate deoxycholate lysed culture. The precipitate was fractionated by size-exclusion chromatography and ion exchange chromatography; therefore, the procedure is complicated and laborious. To streamline the process, a new purification strategy was developed. Briefly, the total cell wall was digested with mutanolysin, lysozyme, and LytA to release the CPS. Proteins were degraded by proteinase K treatment and removed by phenol-chloroform extraction. Digestion fragments and other small molecules were removed by dialysis. The purified CPS was lyophilized, dissolved in heavy water, and analyzed by NMR to compare the purity of the purification scheme with the commercial standard prepared by the traditional method. The procedure can purify CPS with comparable purity (Fig. 4).

Production of ct-Gal epitope

The invention also provides methods to modify purified CPS directly by Ggta-1. Isolated CPS 14 was mixed with UDP-Gal, the cofactor Mn²⁺, and Ggta-1. The modified material was dotted on a nitrocellulose membrane and blotted with lectin IB4 and ECL, which detect a or galactoside, respectively (Fig. 5). Both the total cell wall and purified CPS could be modified by Ggta-1, illustrating the robustness of the glycoengineering scheme.

Amino acid sequences:

> Ggta-1 (A74):

QRNEDESKLKLSDWFNPFKRPEVVTMTKWKAPVVWEGTYNRAVLDNYYAK QKITVGLTVFAVGRYIEHYLEEFLTSANKHFMVGHPVIFYIMVDDVSRMPLIEL GPLRSFKVFKIKPEKRWQDISMMRMKTIGEHIVAHIQHEVDFLFCMDVDQVFQ DKFGVETLGESVAQLQAWWYKADPNDFTYERRKESAAYIPFGEGDFYYHAAI FGGTPTQVLNITQECFKGILKDKKNDIEAQWHDESHLNKYFLLNKPTKILSPEY CWDYHIGLPADIKLVKMSWQTKEYNVVRNNV (SEQ ID NO: 1)

>FutC:

MAFKVVQICGGLGNQMFQYAFAKSLQKHSNTPVLLDITSFDGSNRKMQLELF PIDLPYASAKEIAIAKMQHLPKLVRDALKYMGFDRVSQEIVFEYEPKLLKPSRL TYFYGYFQDPRYFDAISSLIKQTFTLPPPPENGNNKKKEEEYHRKLSLILAAKN SVFAHIRRGDYVGIGCQLGIDYQKKAVEYMAKRVPNMELFVFCEDLKFTQNL DLGYPFMDMTTRDKDEEAYWDMLLMQSCKHGIIANSTYSWWAAYLINNPG KIIIGPKHWLFGHENILCKEWVKIESHFEVKSQKYNA (SEQ ID NO: 2) >HMU_12050 (Codon optimised):

MMQSTAQNTQQNTHFAGSSQTTPQAAQSVQQASLALPKSSPTCYKIAILYICT

GAYSIFWQDFYDSAKVHLLPAHRLTYFVFTDADSLYAEEASDVRKIYQENLG

WPFNTLKRFEMFLGQEEALREFDFVFFFNANCLFFQHIGDEFLPIEEDILVTQH

YGFRDASPECFTYERNPKSLAYVPFGKGKAYVYGSTNGGKAGAFLALARTLQ

ERIQEDLSRGIIAIWHDESHLNAYIIDHPNYKMLDYGYGFPEGYGRVPGGGVYI

FLRDKSRVIDVNAIKGMGSPANRRLKNALRKLKHFSKRLLGR (SEQ ID NO: 3)

>FutT

FQPLLDAFIESASIEKMVSKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRY

AITLHQNPNESSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIG

FDELDFNDRYLRMPLYYAHLHYEAELVNDTTAPYKLKDNSLYALKKPSHHFK

ENHPNLCAVVNDESDLLKRGFASFVASNANAPMRNAFYDALNSIEPVTGGGS

VRNTLGYKVGNKSEFLSQYKFNLCFENSQGYGYVTEKILDAYFSHTIPIYWGS

PSVAKDFNPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLYENPLNTLDG

KAYFYQDLSFKKILDFFKTILENDTIYHNNPFIFYRDLHEPLISIDDLRVNYDDL

RVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDL

RVNYDDLRV (SEQ ID NO: 4)

>CpsK

MIKKIEKDLISVIVPIYNVEDYLVECIESLIVQTYRNIEILLINDGSTDNCATIAKE

FSERDCRVIYIEKSNGGLSEARNYGIYHSKGKYLTFVDSDDKVSSDYIANLYN

AIQKHDSSIAIGGYLEFYERHNSIRNYEYLDKVISVEEALLNMYDIKTYGSIFIT

AWGKLFHKSIFNDLEFALNKYHEDEFFNYKAYLKANSITYIDKPLYHYRIRVG

SIMNNSDNVIIARKKLDVLSALDERIKLITSLRKYSVFLQKTEIFYVNQYFRTKK

FLKQQSVMFKEDNYIDAYRMYGRLLRKVKLVDKLKLIKNRFF (SEQ ID NO: 5)

>WcfB (Codon optimized)

MLYVILRGRLGNNLFQIATAASLTQNFIFCTVNKDQERQVLLYKDSFFKNIKV

MKGVPDGIPYYKEPFHEFSRIPYEEGKDLIIDGYFQSEKYFKRSVVLDLYRITDE

LRKKIWNICGNILEKGETVSIHVRRGDYLKLPHALPFCGKSYYKNAIQYIGEDK

IFIICSDDIDWCKKNFIGKRYYFIENTTPLLDLYIQSLCTHNIISNSSFSWWGAWL NENSNKIVIAPQMWFGISVKLGVSDLLPVSWVRLPNNYTLGRYCFALYKVVE

DYLLNILRLIWKRKKNM* (SEQ ID NO: 6)

>WbgL (Codon optimized)

MSIIRLQGGLGNQLFQFSFGYALSKINGTPLYFDISHYAENDDHGGYRLNNLQI

PEEYLQYYTPKINNIYKLLVRGSRLYPDIFLFLGFCNEFHAYGYDFEYIAQKWK

SKKYIGYWQSEHFFHKHILDLKEFFIPKNVSEQANLLAAKILESQSSLSIHIRRG

DYIKNKTATLTHGVCSLEYYKKALNKIRDLAMIRDVFIFSDDIFWCKENIETLL

SKKYNIYYSEDLSQEEDLWLMSLANHHIIANSSFSWWGAYLGSSASQIVIYPTP

WYDITPKNTYIPIVNHWINVDKHSSC* (SEQ ID NO: 7)

>WbsJ (Codon optimized)

MEVKIIGGLGNQMFQYATAFAIAKRTHQNLTVDISDAVKYKTHPLRLVELSCS

SEFVKKAWPFEKYLFSEKIPHFMKKGMFRKHYVEKSLEYDPDIDTKSINKKIV

GYFQTEKYFKEFRHELIKEFQPKTKFNSYQNELLNLIKENDTCSLHIRRGDYVS

SKIANETHGTCSEKYFERAIDYLMNKGVINKKTLLFIFSDDIKWCRENIFFNNQI

CFVQGDAYHVELDMLLMSKCKNNIISNSSFSWWAAWLNENKNKTVIAPSKW

FKKDIKHDIIPESWVKL* (SEQ ID NO: 8)

Claims

23 CLAIMS

1. A bacterial glycan modified to comprise at least one heterologous antigen.

2. The bacterial glycan of claim 1, wherein the at least one heterologous antigen comprises a mammalian antigen, such as a human or porcine antigen.

3. The bacterial glycan of claim 2, wherein the mammalian antigen comprises an ABO blood group antigen, a Lewis antigen, or an a-Gal antigen.

4. The bacterial glycan of claim 3, wherein the ABO blood group antigen comprises a Fuc(al,2)Gal motif linked to GlcNAc.

5. The bacterial glycan of claim 3, wherein the Lewis antigen comprises a Fuc(al,3)GlcNAc linked to Gal.

6. The bacterial glycan of any one of claims 3 to 5, wherein the ABO blood group antigen is selected from the group consisting of a blood group antigen A, a blood group antigen B, and a blood group antigen H.

7. The bacterial glycan of any one of claims 1 to 6, wherein the bacterial glycan comprises Gal(P 1 ,4)GlcNAc((31 , 3)Gal(P 1 ,4)Glc.

8. The bacterial glycan of any one of claims 1 to 7, wherein the bacterial glycan comprises a paragloboside mimic.

9. The bacterial glycan of claim 8, wherein the bacterial glycan comprises a Streptococcus pneumoniae serotype 14 CPS.

10. The bacterial glycan of claim 8 or 9, wherein the bacterial glycan comprises a repeating unit of the following structure:

The bacterial glycan of any one of claims 1 to 10, wherein the glycan is conjugated to a protein or a lipid. A method of producing a modified bacterial glycan comprising at least one heterologous antigen, the method comprising the step of contacting a bacterial glycan with at least one glycosyltransferase and at least one donor molecule. The method of claim 12, wherein the donor molecule is a monosaccharide derivative. The method of claim 13, wherein the monosaccharide derivative is a nucleotide-activated monosaccharide. The method of any one of claims 12 to 14, wherein the bacterial glycan is modified by the addition of at least one donor molecule to the glycan, through the action of at least one glycosyltransferase, to produce a mammalian antigen in the modified glycan. The method of any one of claims 12 to 15, wherein the glycosyltransferase polypeptide is selected from the group consisting of alpha- 1,3 galactosyltransferase (Ggtal), alpha-1,2 fucosyltransferase (FutC), alpha-1,3 fucosyltransferase (FutT), alpha-2,3 sialyltransferase (CpsK) and alpha-1,3 N- acetylgalactosamine transferase (HMU_12050), or a functional fragment thereof. The method of any one of claims 12 to 16, wherein the monosaccharide is selected from the group consisting of galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), N- acetylneuraminic acid (Sia) and glucose (Glu). The method of any one of claims 12 to 17, wherein the bacterial glycan comprises Streptococcus pneumoniae serotype 14 CPS. The method of claim 18, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with Ggtal and UDP-Gal. The method of claim 18 or 19, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with FutC and GDP-Fuc. The method of claim 18, 19, or 20, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with HMU_12050 and GDP- GalNAc. The method of claim 18, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with FutT and GDP-Fuc. The method of claim 22, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with FutC and GDP-Fuc. The method of claim 22, wherein the method comprises contacting the Streptococcus pneumoniae serotype 14 CPS with CpsK and CMP-NeuNAc^ The method of any one of claims 12 to 24, wherein the method comprises a prior step of isolating the glycan from bacteria. The method of any one of claims 12 to 25, wherein the method further comprises conjugating the modified glycan to a protein. The method of any one of claims 12 to 26, wherein the method further comprises immobilizing the modified glycan onto a surface. 26 A modified glycan obtained according to a method of any one of claims 12 to 27. A conjugate comprising a glycan of any one of claims 1 to 11 or 28 and a carrier protein. A composition comprising a modified CPS of any one of claims 1 to 11 or 28 or a conjugate of claim 29. A method of reducing or eliminating one or more components from a blood product from a subject, the method comprising a) contacting the blood product with a modified glycan of any one of claims 1 to 11 or 28, wherein the modified glycan is immobilized on or within a substrate, so as to remove the one or more components from the blood product. The method of claim 31 , wherein the blood product is whole blood, blood plasma, or completely or partially purified blood plasma. The method of claim 31 or 32, wherein the method comprises the blood product is returned to the subject or is collected for further use following step a). A kit for reducing or eliminating one or more components from a blood product from a subject, the kit comprising a substrate, wherein the substrate comprises a modified glycan of any one of claims 1 to 11 or 28 immobilized on or within the substrate. A method for treating a subject of a disease or condition, the method comprising administering a modified glycan of any one of claims 1 to 11 or 28 or a conjugate of claim 29 to the subject. The method of claim 35, wherein the disease or condition comprises cellular, tissue, or organ transplant rejection or a blood transfusion rejection.