WO2024079210A1 - Protéine de fusion sdab-cbm liée à un polymère de polysaccharide - Google Patents

Protéine de fusion sdab-cbm liée à un polymère de polysaccharide Download PDF

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Publication number
WO2024079210A1
WO2024079210A1 PCT/EP2023/078242 EP2023078242W WO2024079210A1 WO 2024079210 A1 WO2024079210 A1 WO 2024079210A1 EP 2023078242 W EP2023078242 W EP 2023078242W WO 2024079210 A1 WO2024079210 A1 WO 2024079210A1
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composition
sdab
cbm
pharmaceutical
dietary
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PCT/EP2023/078242
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English (en)
Inventor
Mads Laustsen
Signe Christensen
Sandra WINGAARD THRANE
Ágnes DUZS
Christel JØRGENSEN
Andreas HOUGAARD LAUSTSEN-KIEL
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Bactolife A/S
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Publication of WO2024079210A1 publication Critical patent/WO2024079210A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to a composition
  • a composition comprising a fusion protein comprising a single domain antibody (sdAb) and a carbohydrate-binding module (CBM) bound to a polysaccharide polymer via a not covalent binding of the polysaccharide polymer to the CBM (termed “sdAb-CBM:pSac”) - and use of it in the prevention or treatment of a pathogen induced infection in the gastrointestinal tract.
  • sdAb single domain antibody
  • CBM carbohydrate-binding module
  • sdAb refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.
  • Single domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco.
  • Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian host cell).
  • sdAbs single domain antibodies
  • common antibodies which are composed of two heavy protein chains and two light chains.
  • Nanobodies have been shown to be just as specific as antibodies, and in some cases, they are more robust. The smaller size and single domain make these antibodies easier to transform into microorganism (e.g. bacteria) cells for bulk production.
  • microorganism e.g. bacteria
  • WO2020/144164A1 (Bactolife) describes use of sdAbs for the treatment of a pathogen induced infection in the gastrointestinal (Gl) tract of an animal (e.g. a pig, a cattle, etc) or human subject.
  • Gl gastrointestinal
  • sdAb monomers as such and divalent sdAb-linker-sdAb constructs are described - in e.g. Example 13 is shown that divalent sdAb-linker-sdAb constructs may work better than corresponding sdAb monomers.
  • a pathogen induced infection may be caused by the pathogen itself (e.g. a bacteria such as e.g. E. coli) or be caused by a molecule (e.g. a toxin) produced by the pathogen (such as e.g. toxin B produced by the pathogen Clostridium difficile).
  • the pathogen itself
  • a molecule e.g. a toxin
  • the pathogen such as e.g. toxin B produced by the pathogen Clostridium difficile
  • CBMs carbohydrate-binding modules
  • CBMs were classified as cellulose-binding domains (CBDs), because the first examples of these classes of protein domains bound to crystalline cellulose.
  • the article of Hussack et al. (Sensors 2009, 9, 5351-53) describes an immobilization platform for sensing of pathogens, biomarkers and environmental pollutants - which comprises sdAb attached to CBM and linked to a cellulose support.
  • the article Barbosa et al. (Acta Biomaterialia 143 (2022) 216-232) describes peptide antimicrobial cellulose-based materials (e.g. hydrogels, paper) for applications such as wound dressings, tissue scaffolds, coatings.
  • peptide antimicrobial cellulose-based materials e.g. hydrogels, paper
  • a fusion protein comprising a single domain antibody (sdAb) and a carbohydrate-binding module (CBM) bound to a polysaccharide polymer via a not covalent binding of the polysaccharide polymer to the CBM (termed “sdAb-CBM:pSac”) for the prevention/treatment of a pathogen induced infection in the gastrointestinal tract of an animal/human subject.
  • sdAb single domain antibody
  • CBM carbohydrate-binding module
  • a problem to be solved by the present invention relates to the provision of a novel pharmaceutical/dietary composition/combination that advantageously may e.g. be used for the prevention/treatment of a pathogen induced infection in the gastrointestinal tract (Gl) of an animal (e.g. a pig, a cattle, etc) or human subject - such as e.g. reducing virulence in the Gl and thereby stabilizing the healthy microbiome.
  • Gl gastrointestinal tract
  • composition comprising a compound, which is a fusion protein comprising a single domain antibody (sdAb) and a carbohydrate-binding module (CBM) bound to a polysaccharide polymer via a not covalent binding of the polysaccharide polymer to the CBM (termed “sdAb-CBM:pSac”) and wherein the composition is:
  • sdAb-CBM refers to a covalent binding - something that skilled person understands relates to that it is a fusion protein.
  • CBM:pSac refers to a not covalent binding - as known in the art CBM binds to carbohydrate (e.g. a polysaccharide polymer) via not covalent binding.
  • Figure 1 herein shows an illustrative example of a sdAb-CBM:pSac (termed sdAb-CBM:polymer in the figure) compound compared to WO2020/144164A1 (Bactolife) prior art divalent sdAb-linker-sdAb constructs.
  • the sdAb binds to a toxin molecule produced by the pathogen.
  • results of this example show a surprisingly high improved sdAb resistance against gastric and intestinal proteases for the sdAb-CBM:pSac compound - this is of course advantageous for the treatment of a pathogen induced infection in the gastrointestinal tract of an animal/human.
  • results of this example show a surprisingly high/strong binding of sdAb-CBM fusion protein to cellulose at pH 1 .2 - i.e. sdAb-CBM:pSac compound may function at this low pH - i.e. it may function at the gastric/stomach acid fluid with a pH between 1 and 3.
  • CBMs are small components of several enzymes - i.e. the skilled person would prima facie expect they work best at optimal activity pH values of the relevant natural enzymes.
  • results of this example demonstrate a surprisingly good capacity of sdAb-CBM:pSac compounds of the invention to remove bacteria at even at low bacteria concentration - which evidently is of significant clinical relevance - since it e.g. gives the possibility to intervene early in the course of a bacterial infection (e.g. for prevention and/or prophylaxis related treatment), where the bacteria concentration generally is low.
  • composition comprising numerous individual sdAb-CBM :pSac compounds would be suitable for use in the treatment of a pathogen induced infection in the gastrointestinal tract of an animal/human subject.
  • an advantage of the sdAb-CBM :pSac compound as discussed herein could be, that it as a compound as such may be able to bind a higher number of pathogens/toxins as compared to e.g. a sdAb-linker-sdAb construct of the prior art - something that may be an advantage for removal of pathogens/toxins present in low amounts in the gastrointestinal tract (see e.g. Example 5 herein).
  • WO2020/144164A1 Bactet al.
  • crosslinking of pathogens may be important for removal pathogens - e.g. to create a bigger structure that relatively easily may be removed from the gastrointestinal tract.
  • an advantage of the sdAb-CBM:pSac compound could be that less crosslinking of pathogens/toxins may be required for e.g. proper clearance/removal, since the sdAb- CBM:pSac compound is by itself relatively big and may therefore relatively easily be removed from the gastrointestinal tract - something that e.g. could be an advantage if pathogens/toxins are present in low amounts and it may thereby be statistically difficult to crosslink sufficient pathogens/toxins in order to get a sufficient big molecule that relatively easily may be removed from the gastrointestinal tract.
  • crosslinking should be relevant for removal of a particular pathogens/toxins - sdAb-CBM:pSac compound as discussed herein may comprise numerous sdAb bindings sites - i.e. may be very useful for relatively rapidly get crosslinking of many pathogens/toxins.
  • a first aspect of the present invention relates to a pharmaceutical or dietary composition
  • a pharmaceutical or dietary composition comprising a compound, which is a fusion protein comprising a single domain antibody (sdAb) and a carbohydrate-binding module (CBM) bound to a polysaccharide polymer via a not covalent binding of the polysaccharide polymer to the CBM (termed “sdAb-CBM:pSac”) and wherein the composition is: (i): comprising from 10 3 to 10 20 of individual sdAb-CBM:pSac compounds per gram(g) weight of the composition as such; and wherein the sdAb binds at least one pathogen surface component and/or at least one molecule produced by a pathogen.
  • sdAb single domain antibody
  • CBM carbohydrate-binding module
  • the term “individual” in relation to the term “individual sdAb-CBM:pSac compounds” of the first aspect should be understood as the skilled person would understand it in the present context - i.e. that it refers to that there is no significant binding between the individual sdAb-CBM:pSac compounds.
  • the individual sdAb-CBM:pSac compounds of the composition may then actively work at different places of the gastric system and thereby remove pathogens/toxins at different places of the gastrointestinal (Gl) tract - i.e. thereby obtain treatment of a pathogen induced infection at different places of the gastrointestinal tract.
  • pathogen relates to any organism or agent that can produce a disease - wherein the term disease is understood broadly to cover e.g. a pathogen induced infection in the gastrointestinal tract of an animal or human subject - i.e. the disease is preferably related to a pathogen induced infection in the gastrointestinal tract of an animal or human subject.
  • CBM/CBD based immobilization on a purification column is not related to a pharmaceutical or dietary composition of the first aspect.
  • CBM may bind to a polysaccharide polymer with a relatively high binding efficiency - accordingly, the sdAb-CBM and polysaccharide polymer may be administered to an animal or human subject of interest as a pharmaceutical combination for e.g. separate or sequential medical use.
  • sdAb-CBM fusion proteins and polysaccharide polymer are administered as two separate compositions, then it is believed that enough sdAb-CBM proteins and polysaccharide polymers would be able to find each other in the gastrointestinal tract in order to in vivo create sufficient number of sdAb-CBM:pSac compounds to get a herein relevant prevention/treatment of a pathogen induced infection in the gastrointestinal tract of an animal or human subject.
  • a pharmaceutical or dietary composition of the first aspect is administered to an animal or human subject.
  • a second aspect of the present invention relates to pharmaceutical or dietary combination comprising:
  • sdAb single domain antibody
  • CBM carbohydrate-binding module
  • the term "combination" of the second aspect relates herein to the various combinations of (A) and (B), for example in a single pharmaceutical/dietary composition or in separate compositions.
  • the order of applying (A) and (B) is not essential.
  • a combination of (A) and (B) can be formulated for its simultaneous, separate or sequential administration.
  • the compositions (A) and (B) are administered in a relatively close time proximity to each other.
  • a pathogen induced infection may be caused by the pathogen itself (e.g. a bacteria such as e.g. E. coli) or be caused by molecule (e.g. a toxin) produced by the pathogen (such as e.g. toxin B produced by the pathogen Clostridium difficile).
  • the pathogen itself
  • molecule e.g. a toxin
  • the pathogen such as e.g. toxin B produced by the pathogen Clostridium difficile
  • prevention or treatment of a pathogen induced infection in the gastrointestinal tract should be understood as the skilled person would understand it in the present context - i.e. to broadly cover any herein relevant prevention/treatment of a pathogen induced infection - such as e.g. removal, clearance, blocking and/or inactivating the pathogens/toxins or e.g. reducing risk of infection in the sense that the pathogens do not colonize in the gastric system or e.g. reducing virulence in the Gl and thereby stabilizing the healthy microbiome or e.g. so-called microbiome engineering, where e.g. number of unwanted (e.g.
  • pathogens e.g. E. coli bacteria
  • IBD inflammatory bowel disease
  • an unpleasant condition due to an unbalanced microbiome e.g. an unpleasant condition due to an unbalanced microbiome
  • Figure 1 Illustration of an example of sdAb-CBM:pSac compound as discussed herein compared to WO2020/144164A1 (Bactolife) prior art divalent sdAb-linker-sdAb constructs.
  • composition of interest is pharmaceutical or dietary composition or not - i.e. it comprises pharmaceutical or dietary acceptable components.
  • the pharmaceutical or dietary composition may comprise different types of sdAb, CBM and/or pSac.
  • the composition may comprise different types of sdAbs binding to e.g. different pathogens/toxins of interest.
  • the composition may comprise some individual sdAb-CBM:pSac compounds, where pSac is e.g. a cellulose polymer and other individual sdAb-CBM:pSac compounds, where pSac is e.g. a starch polymer.
  • the pharmaceutical or dietary composition may be coated - for instance with an enteric coating to prevent dissolution or disintegration in the gastric environment, which may be preferred it main target of the composition is the intestine.
  • composition all components (alternatively termed excipients) of the composition should be pharmaceutically acceptable.
  • pharmaceutically acceptable we mean a non-toxic material that does not significantly decrease the effectiveness of the sbAb.
  • Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)).
  • the pharmaceutical composition may further comprise one or more further active components such as e.g. an antibiotics, fecal matter transfer and/or monoclonal antibodies.
  • further active components such as e.g. an antibiotics, fecal matter transfer and/or monoclonal antibodies.
  • the pharmaceutical composition may e.g. be a tablet, a gel, a suspension, a solution, a liposome, a capsule, a powder, or encapsulated in polymers - preferably, the pharmaceutical composition is a tablet, a capsule or a powder.
  • composition For a dietary composition, all components (alternatively termed excipients) of the composition should be dietarily acceptable.
  • the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and/or nutrients, such as dietary minerals.
  • the dietary composition is a food, a feed, or a beverage - or a food additive, a feed additive, or a beverage additive - or food ingredient, a feed ingredient, or a beverage ingredient - or food supplement, a feed supplement, or a beverage supplement.
  • the dietary composition may e.g. be a pellet (preferably when the dietary composition is a feed), tablet, a gel, a suspension, a solution, a liposome, a capsule, a beverage, a powder, or encapsulated in polymers - preferably, the dietary composition is a pellet (preferably when the dietary composition is a feed), tablet, a capsule, a beverage or a powder.
  • the dietary composition is a pellet composition.
  • the dietary composition is a tablet or a powder - which can e.g. be used as a beverage supplement.
  • the dietary composition may e.g. be a so-called Medical Devise composition - such as e.g. a Medical Devise composition in accordance with e.g. European Union (EU) Medical Devices Regulation (MDR) 2017/745 - for instance a so-called class II medical devise according to e.g. the MDR.
  • a so-called Medical Devise composition - such as e.g. a Medical Devise composition in accordance with e.g. European Union (EU) Medical Devices Regulation (MDR) 2017/745 - for instance a so-called class II medical devise according to e.g. the MDR.
  • EU European Union
  • MDR Medical Devices Regulation
  • the dietary composition may e.g. be a so-called medical food composition.
  • the pharmaceutical or dietary composition of the first aspect and/or embodiment thereof is a composition with a weight of the composition as such of at least 100g, more preferably least 200g, even more preferably least 500g, and most preferably at least 1 kg.
  • sdAb-CBM refers to a covalent binding - something that skilled person understands relates to it is a fusion protein.
  • the sdAb-CBM covalent binding does not have to be a direct covalent binding between sdAb and CBM.
  • the preferred linker is a glycine serine linker of the structure (G x S) n , where x may be a number between 1 to 50, preferably between 1 to 25, more preferably between 1 to 10, most preferably between 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 50, preferably between 1 to 25, more preferably between 1 to 10, most preferably between 2 to 5.
  • the sdAb-CBM fusion protein may comprises more than one sdAb - such as e.g. two or more sdAbs
  • the fusion protein may also comprise other elements of interest - such as e.g. polypeptide/protein sequences that e.g. could facilitate purification.
  • the discussed sdAb-CBM fusion proteins of working examples herein are all examples of sdAb-CBM fusion proteins, where sdAb and CBM are bound together via covalent peptide bonds.
  • the sdAb-CBM fusion protein of the first and/or second aspect and/or embodiment thereof is a sdAb-CBM fusion protein, wherein the sdAb-CBM fusion protein is a polypeptide sequence (preferably recombinantly expressed) and wherein the sdAb and CBM are bound together via covalent peptide bonds - e.g. directly or e.g. via a suitable amino acid linker sequence between the sdAb and CBM.
  • sdAb refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.
  • Such single domain antibodies can be derived from antibodies raised in e.g. sharks or e.g. Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco.
  • Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian hoist cell).
  • Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • the single domain antibodies of the disclosure can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab,” as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins,
  • a sdAb may comprise relevant complementary-determining regions - i.e. in a preferred embodiment, the sdAb is comprising:
  • CDR1 complementary-determining region 1
  • SEQ ID NO: i SEQ ID NO: i
  • CDR2 complementary-determining region 2
  • a complementary-determining region 3 comprising or consisting of a relevant sequence (may e.g. be termed SEQ ID NO: iii).
  • Carbohydrate-binding module (CBM)
  • CBMs carbohydrate-binding modules
  • CBMs were classified as cellulose-binding domains (CBDs), because the first examples of these classes of protein domains bound to crystalline cellulose.
  • the skilled person knows or may routinely determine - if protein of interest comprises a CBM - i.e. may routinely determine if a fusion protein of interest is a sdAb-CBM fusion protein or not.
  • cited Oliveira review article describes that CBMs can be divided into different so-called fold families (se e.g. Table 1 of the Oliveira review article) - i.e. the skilled person may based on e.g. the herein cited Oliveira review article and relevant common general knowledge routinely determine whether a CBM of interest belongs to a specific fold family (e.g. fold family 3) or not.
  • fold families e.g. Table 1 of the Oliveira review article
  • relevant common general knowledge routinely determine whether a CBM of interest belongs to a specific fold family (e.g. fold family 3) or not.
  • the CBM belongs to any of the fold families 1-3 - more preferably the CBM belongs to any of the fold families 1 or 3 - even more preferably the CBM belongs to the fold family 3.
  • the preferred choice of a specific CBM relates to the preferred polysaccharide polymer of interest (see below) - i.e. if the polysaccharide polymer of interest is a cellulose polymer then shall the suitable CBM be able to bind the cellulose polymer.
  • a preferred CBM may be a type A CBM.
  • CBM comprising SEQ ID NO: 3 or SEQ ID NO: 4 - accordingly, it may be preferred that the CBM is comprising SEQ ID NO: 3 or SEQ ID NO: 4.
  • polysaccharides are chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages.
  • the polysaccharide polymer may be linear or branched.
  • the polysaccharide polymer may be a homopolysaccharide or a heteropolysaccharide.
  • the polysaccharide polymer may comprise not monosaccharide structures/elements and still be understood to be as herein suitable polysaccharide polymer.
  • CBM carbohydrate-binding module
  • the skilled person may routinely determine the preferred numbers of monomers in a polysaccharide polymer of interest - but preferably it should at least longer than 9 monomers.
  • the polysaccharide polymer comprises from 10 to 50000 monomers, more preferably from 10 to 10000 monomers, even more preferably from 10 to 8000 monomers, and most preferably from 50 to 1000 monomers.
  • the polysaccharide polymer is a cellulose polymer, a derivative of a cellulose polymer, a hemicellulose polymer, a derivative of a hemicellulose polymer, a starch polymer, or a derivative of a starch polymer.
  • a derivative is a compound that is derived from a similar compound by a chemical reaction.
  • - chitosan may be considered as a derivative of chitin and both chitosan and chitin are polysaccharide polymers.
  • a derivative of a polysaccharide polymer of interest e.g. a derivative of a cellulose polymer
  • the skilled person may based on common general knowledge routinely determine whether a polysaccharide polymer of interest is a herein relevant derivative or not - such as e.g. a derivative of a cellulose polymer.
  • the polysaccharide polymer is a cellulose polymer, a hemicellulose polymer, or a starch polymer - more preferably a cellulose polymer or a hemicellulose polymer - even more preferably a cellulose polymer.
  • a preferred example of a hemicellulose polymer is an arabinoxylan polymer.
  • polysaccharide polymer is plant-based/derived polysaccharide.
  • the polysaccharide polymer (e.g. preferably cellulose polymer) may be a crystalline or amorphous polymer - it may be preferred that the polysaccharide polymer (e.g. preferably cellulose polymer) is an amorphous polymer.
  • MMC microcrystalline cellulose
  • the polysaccharide polymer e.g. preferably cellulose polymer
  • the polysaccharide polymer is an crystalline polymer - such as preferably a microcrystalline cellulose (MCC) polymer.
  • the polysaccharide polymer is a crystalline cellulose polymer (preferably MCC) if the CBM belongs to any of the fold families 1 or 3 (even more preferably if the CBM belongs to the fold family 3).
  • the polysaccharide polymer may be added to the cell lysate (or supernatant) comprising the expressed sdAb-CBM fusion protein and the sdAb-CBM:pSac compounds may then be created/obtained by not covalent binding of the polysaccharide polymer to the CBM.
  • the sdAb-CBM:pSac compounds may be obtained in relatively pure form by e.g. simple centrifugation or more sophisticated known purification technology. As discussed above in relation to e.g. a pharmaceutical/dietary combination of the second aspect - if sdAb-CBM fusion proteins and polysaccharide polymer are administered as two separate compositions then it is believed that enough sdAb-CBM proteins and polysaccharide polymers would be able to find each other in the gastrointestinal tract in order to in vivo create sufficient of sdAb-CBM:pSac compounds to get a herein relevant the prevention/treatment of a pathogen induced infection in the gastrointestinal tract of an animal or human subject.
  • the first aspect relates in point (i) to that the composition is comprising from 10 3 to 1 O 20 of individual sdAb-CBM :pSac compounds per gram(g) weight of the composition as such.
  • the composition is comprising from 10 5 to 1 O 20 of individual sdAb-CBM:pSac compounds per gram(g) weight of the composition as such, more preferably the composition is comprising from 10 5 to 10 19 of individual sdAb-CBM:pSac compounds per gram(g) weight of the composition as such, even more preferably the composition is comprising from 10 6 to 10 19 of individual sdAb-CBM:pSac compounds per gram(g) weight of the composition as such; and most preferably the composition is comprising from 10 1 ° to 10 18 of individual sdAb-CBM:pSac compounds per gram(g) weight of the composition as such.
  • composition of a herein relevant aspect may e.g. be suspension/solution.
  • 1 ml of an aqueous solution has a weight of close to 1 mg.
  • composition of a herein relevant aspect may comprise different types of sdAb-CBM:pSac compounds - for instance with different types of sdAbs directed to different types of pathogen.
  • the composition of the first aspect may comprise 10 5 /g of sdAb-CBM:pSac compounds with sdAb directed to (i.e. binding) E. coli bacteria pathogens and also 10 5 /g of sdAb-CBM:pSac different compounds with sdAb directed to (i.e. binding) another bacteria pathogens - such as composition then comprises 2x10 5 /g sdAb-CBM:pSac compounds.
  • a preferred sdAb-CBM:pSac compound may e.g. be wherein the compound comprises more than one sdAb-CBM bound to the polysaccharide polymer (see e.g. Figure 1 for an illustrative example).
  • sdAb-CBM:pSac compound wherein the compound comprises more than one sdAb- CBM bound to the polysaccharide polymer - it may be preferred that the more than one (i.e. two or more, such as 5 or more or such as 10 or more) sdAb-CBMs bound to the polysaccharide polymer are comprising different types of sdAbs (e.g. one sdAb-CBM comprises one type of sdAb and another sdAb-CBM comprises a different type of sdAb) - accordingly the sdAb-CBM:pSac compound will comprise different types of sdAbs that e.g. could bind to different types of pathogens or bind to different epitopes of the same pathogen. sdAb bind pathogen or at least one molecule produced by a pathogen
  • the herein relevant aspects relate to that the sdAb binds at least one pathogen surface component and/or at least one molecule produced by a pathogen.
  • the pathogen is an enterotoxic pathogen and/or at least one molecule (preferably an enterotoxin) produced by an enterotoxic pathogen.
  • an enterotoxin is a toxin released by a pathogen (e.g. a microorganism) that targets the intestine - i.e. an enterotoxic pathogen (preferably an enterotoxic bacterium or an enterotoxic virus) is a pathogen that targets the intestine.
  • a pathogen e.g. a microorganism
  • an enterotoxic pathogen preferably an enterotoxic bacterium or an enterotoxic virus
  • the pathogen is a bacterium, a virus, a fungus, a protozoa and/or a worm - more preferably the pathogen is a bacterium or a virus (e.g. a rotavirus or a norovirus).
  • the pathogen is an Escherichia coli bacteria, a Vibrio cholera bacteria, a Salmonella, a Campylobacter bacteria, a Staphylococcus bacteria, a Listeria bacteria, a Shigella bacteria, Mycoplasma and/or Clostridium difficile.
  • the molecule produced by a pathogen is a toxin, an inhibitor and/or an enzyme, such as betalactamase - more preferably, the molecule produced by a pathogen is a toxin.
  • the toxin is toxin B produced by the pathogen Clostridium difficile - see e.g. EP21211996.0 (Bactolife - not published at filing/priority date of present application - does e.g. not mention anything of relevance in relation to e.g. CBM).
  • sdAb comprising SEQ ID NO: 1 - which may be a preferred sdAb.
  • the sdAb comprising SEQ ID NO: 1 binds to a toxin produced by the pathogen Clostridium difficile.
  • Example 3 essentially demonstrate that it is possible in a relatively cheap/rapid way to get a composition comprising sdAb-CBM:pSac compounds with very little (if any measurable) amount of DNA contamination.
  • the method to remove DNA may be seen as based on that the inventors found that surprisingly little (if any measurable) amount of DNA binds to sdAb-CBM:pSac compounds, and these sdAb-CBM:pSac compounds relatively easy precipitate in a liquid culture medium used for e.g. recombinant expression based production.
  • EU European Union
  • the composition of first aspect (preferably wherein the composition is a dietary composition) comprises less than 15 ng DNA per gram(g) weight of the composition as such - more preferably, the composition comprises less than 10 ng DNA per gram(g) weight of the composition as such - most preferably, the composition comprises less than 5 ng DNA per gram(g) weight of the composition as such.
  • the European Food Safety Authority requires no detection of DNA by a method with a detection limit of 10 ng or below (DOI: 0.2903/j.efsa.2021 .6851).
  • the second aspect relates to pharmaceutical or dietary combination comprising:
  • sdAb single domain antibody
  • CBM carbohydrate-binding module
  • compositions (A) and (B) are e.g. two separate compositions - then will the preferred amount of sdAb-CBM fusion proteins in (A) generally depend on the amount polysaccharide polymers in (B) - if there is e.g. 10 6 polysaccharide polymers in (B) then it will normally be preferred that there is at least 10 6 sdAb-CBM fusion proteins in (A).
  • composition (B) comprising polysaccharide polymers may e.g. be mixed with the composition (A) comprising “sdAb-CBM” protein before given to the animal or human subject of interest.
  • the composition (A) is comprising from 10 5 to 1 O 20 of individual sdAb-CBM per gram(g) weight of the composition as such, more preferably the composition is comprising from 10 5 to 10 19 of individual sdAb-CBM per gram(g) weight of the composition as such, even more preferably the composition is comprising from 10 6 to 10 19 of individual sdAb-CBM per gram(g) weight of the composition as such; and most preferably the composition is comprising from 10 1 ° to 10 18 of individual sdAb-CBM per gram(g) weight of the composition as such.
  • the pharmaceutical combination is a single composition comprising both Compound (A) and Compound (B) - such as most preferably, wherein the second aspect relates to a pharmaceutical or dietary composition of the first aspect and/or embodiment thereof, for use in the prevention or treatment of a pathogen induced infection in the gastrointestinal tract of an animal or human subject
  • the gastrointestinal tract is intestine - i.e. a prevention or treatment of a pathogen induced infection in the intestine of an animal or human subject.
  • WO2020/144164A1 Bactet al.
  • sdAbs for the treatment of a pathogen induced infection in the gastrointestinal (Gl) tract of an animal (e.g. a pig, a cattle, etc) or human subject.
  • the animal is a pig, a cattle, a poultry, a sheep, a goat, a horse, a chicken, a donkey, a mule, a duck, a geese or a turkey - more preferably the animal is a pig, a cattle or a poultry - most preferably the animal is a pig.
  • the composition is administered enterally - such as orally, such as a food supplement, as a tablet, a gum (e.g. a gummy bear) or a gel, or via gastric intubation.
  • enterally such as orally, such as a food supplement, as a tablet, a gum (e.g. a gummy bear) or a gel, or via gastric intubation.
  • the term “enterally” relates administration via the gastrointestinal tract.
  • the prevention or treatment is for reducing virulence in the Gl and thereby stabilizing the healthy microbiome - in particular, when the composition is a dietary composition.
  • a separate aspect of the invention - a method to remove DNA
  • a separate aspect of the invention relates to a method to obtain a composition
  • a composition comprising a compound, which is a fusion protein comprising a protein (Prot) and a carbohydrate-binding module (CBM) bound to a polysaccharide polymer via a not covalent binding of the polysaccharide polymer to the CBM (termed “Prot-CBM:pSac”) and wherein the composition is:
  • the recombinant expressed Prot-CBM compounds are secreted to the culture medium in step (A) - then may a significant amount of host cell be removed by e.g. centrifugation to get sample (B).
  • Prot-CBM compounds are not secreted (e.g. if E. coli is used as host cell) - cell lysis will generally be required.
  • step (C) The skilled person may routinely determine how much pSac is preferably added in step (C) - it will generally depend on the number of expressed Prot-CBM compounds.
  • Precipitating the Prot-CBM:pSac compounds in step (D) may routinely be done by e.g. centrifugation or other suitable routine techniques.
  • Step (E) is a routine step essentially relating to separating liquid comprising DNA from the precipitated Prot-CBM:pSac compounds - as known in the art, this may e.g. be done by washing (with e.g. water or a suitable aqueous buffer/solution).
  • the protein (Prot) in the DNA removal method of the separate aspect is a single domain antibody (sdAb).
  • step (E) there is in step (E) removed so much DNA that one gets (e.g. after other optional relevant steps - such as relevant up-concentration steps) a composition that comprises less than 15 ng DNA per gram(g) weight of the composition as such - more preferably, the composition comprises less than 10 ng DNA pergram(g) weight of the composition as such - most preferably, the composition comprises less than 5 ng DNA per gram(g) weight of the composition as such.
  • An advantage of the method of the separate aspect relates to that it is not necessary to use DNase and/or column-based purification steps to remove the DNA - accordingly, a preferred embodiment of the method of the separate aspect relates to a method, wherein the method does not involve use of DNase and/or column-based purification steps.
  • composition of the first aspect e.g. preferred CBM, preferred pSac, etc.
  • DNA removal method of the separate aspect e.g. DNA removal method of the separate aspect
  • EXAMPLE 1 Binding to cellulose enhances resistance against gastric and intestinal proteases
  • SEQ ID NO: 1 is identical to SEQ ID NO: 27 of EP21211996.0 (Bactolife - not published at filing/priority date of present application) sdAb’.
  • the sdAb in the sdAB-CBM fusion proteins B004_0005 and B004_0006 as well as both sdAb domains in the sdAb-sdAb fusion protein B003_0005 comprises SEQ ID NO: 1 .
  • CBM CBM’.
  • Two CBMs were used the CBM domain from T. reesei Cellobiohydrolase 11 , cel7a (B004_0005) and T. reseei Cellobiohydrolase II, cel6a (B004_0006). See table above.
  • Polysaccharide polymer Cellulose polymer Avicel pH 101 - overall ranging from ⁇ 100 to 400 glucose monomers - from provider Sigma Aldrich.
  • the sdAb-CBM and sdAb-sdAb fusion proteins was cloned into the pSANGI 0-3F vector (Martin, 2006) ensuring C-terminal 6xHis-tag and 3xFLAG tag.
  • Proteins were expressed in an E. coll strain in autoinduction media (Studier, 2005). After lysis and extraction of periplasmic fraction (Ghamghami, 2020) proteins were purified using Immobilized-metal affinity chromatography (IMAC).
  • IMAC Immobilized-metal affinity chromatography
  • the sdAb-CBM:pSac compounds of the composition were assembled by incubating Avicel PH-101 and sdAb-CBM (4 pmol sdAb-CBM per 1 g of Avicel PH-101) with end-over-end rotation at room temperature for 1 hour.
  • Test of protease resistance Resistance towards proteases was investigated by mixing sdAb-CBM:pSac, sdAb-CBM or sdAb-sdAb with 35 mM NaCI, 0.07 HCI, 200U/L pepsin pH 2 (simulated gastric fluid (SGF)) or 50 mM K3PO4, 10 mg/mL pancreatin pH 6.8 (simulated intestine fluid (SIF)).
  • SGF gastric fluid
  • SIF simulated intestine fluid
  • the table below shows remaining full-length protein (in % of full length protein detected before treatment) after treatment with SGF or SIF.
  • the compound sdAb-CBM:polymer is approximately 2-folds more resistant towards the gastric protease pepsin than sdAb-CBM and at least 46-fold more resistant than a sdAb-sdAb fusion after 10 min of treatment with SGF. Even more surprising, after 2 hours of exposure to a simulated gastric environment full length sdAb-CBM was still present in the sdAb-CBM:polymer composition despite all sdAb-sdAb was degraded after only 10 min as well as minimum 98 % of sdAb-CBM was degraded after 30 min in a composition not comprising a carbohydrate polymer.
  • the sdAb-CBM:polymer composition exhibits ⁇ 2 fold increased resistance towards pancreatin in SIF after 10 min. And, similarly to treatment with SGF detectable levels of sdAb-CBM is present after 120 min of treatment with SIF when in a sdAb-CBM:polymer composition, whereas no full-length sdAb-CBM is detected after 30 min when not associated with a carbohydrate. Similarly, very little, if any, sdAb-sdAb is detected after 30 min treatment with SIF and none after 120 min.
  • the assembly of the sdAb-CBM:polymer is essential for the higher resistance towards proteases. This is evident by the observation that a composition comprising sdAb-sdAb and Avicel PH-101 does not exhibit increased resistance towards proteases.
  • sdAbs have high physicochemical stability as well as resistance against proteases (Harmsen, 2007, Asaadi 2021). Hence, it was highly surprising to the present inventors that the compound (sdAb-CBM :pSac) showed increased resistance to SGF and SIF both short term (10 min) and over an increased period of time (120 min).
  • results of this example show a surprisingly high improved sdAb resistance against gastric and intestinal proteases for the sdAb-CBM:pSac compound - this is of course advantageous for the treatment of a pathogen induced infection in the gastrointestinal tract of an animal/human.
  • EXAMPLE 2 The sdAb-CBM fusion protein binds cellulose at high temperature and low pH
  • Periplasmic fraction from lysed cells were incubated with Avicel PH-101 (1 g Avicel PH-101 per 100 mL culture) and incubated at room temperature with end-to-end rotation for 1 hour to assemble sdAb- CBM:polymer composition.
  • sdAb-CBM:polymer was washed with 150 mM NaCI (10 mL per gram of Avicel PH-101).
  • sdAb-CBM-polymer was treated with 10 mL HCI (pH 1.2) per gram of Avicel PH-101.
  • Absorption of sdAb-CBM to Avicel PH-101 was evaluated by SDS-PAGE before and after treatment.
  • results of this example show a surprisingly high/good binding of sdAb-CBM fusion protein to cellulose at pH 1 .2 - i.e. sdAb-CBM:pSac compound may function at this low pH - i.e. it may function at the gastric/stomach acid fluid with a pH between 1 and 3.
  • EXAMPLE 3 Binding to cellulose reduces DNA contamination when expressed in E. coll
  • sdAb-CBM was eluted by 5 M urea and desalted using PD-10 column (Cytiva).
  • DNAse I (Applichem Lifescience) was performed with 117 U DNase I (brand) per 100 pL cell lysate, avicel-elute or Ni-NTA elute. DNA content was evaluated by gel electrophoresis on 1 % agarose. Results
  • Figure 2 shows that the DNA content in a sdAb-CBM composition expressed in E. coli is reduced significantly by binding to Avicel PH-101 compared with treatment with the nuclease DNAse I. Further, the DNA content after binding to Avicel PH-101 is similar to binding to Ni-NTA beads highly specific for His-tagged proteins.
  • sdAb-CBM and sdAb-sdAB sequences and protein production as in example 1 . Assembly of sdAb- CB:polymer as in example 1 , but with 1.5 pmol sdAb-CBM per gram of avicel. Antigen (Uniprot ID: P18177.3 residue 1-543) was inserted in modified pSANGI O without FLAG-tag. Expression and purification of antigen as proteins in example 1.
  • sdAb-CBM:polymer, sdAb-CBM or sdAb-sdAB was incubated with antigen in 3 times molar excess (in relation to binding site) with end-over-end rotation at room temperature for 2 h.
  • antigen:sdAb-CBM and antigen :sdAb-sdAb:antigen complex were pulled down by centrifugation by addition of anti-FLAG beads (insoluble fraction).
  • Antigen:sdAb-CBM:polymer (insoluble fraction) complex was pulled down by centrifugation. Bound antigen (in insoluble fraction) as fraction of total antigen was evaluated by SDS-PAGE.
  • Table above shows that 98 % of available antigen is bound to a sdAb-CBM:polymer composition compared to 76 % for sdAb-CBM not bound to a carbohydrate polymer and 82 % for sdAb-sdAb. This indicates that assemple of the sdAb-CBM:polymer composition does not interfere with antigen binding. And, at best, might increase antigen binding compared to a sdAb-sdAb composition.
  • sdAb-sdAb fusion protein or “sdAb-sdAb”.
  • composition sdAb’.
  • the sdAb in the sdAB-CBM fusion proteins B004_0011 and B004_0010 as well as both sdAb domains in the sdAb-sdAb fusion protein B001_0003 comprises SEQ ID NO: 5.
  • SEQ ID NO: 5 is known in the art to be a sdAb binding to E. Coll and the Fiil et al. (iScience 25, 104003 April 15, 2022) article reads: “K922 (GenBank accession number: AJ810819)”. CBM’. The same cel6a and cel7a as used in Example 1.
  • Polysaccharide polymer Cellulose polymer Avicel pH 101 - see Example 1 .
  • the sdAb-CBM:pSac (Invention) compounds of the invention are termed B004_0011 :Avicel and B004_0010:Avicel.
  • the “prior art” comparative sdAb-sdAb compound is termed B001_0003.
  • Bacteria:sdAb-CBM:polymer or bacteria:sdAb-sdAb:bacteria complex were pulled down by centrifugation at 50 RCF for 30 seconds. Representative fraction of supernatant was cultured overnight on LB agar plates at 30 °C to quantify CFU per ml.
  • the used bacteria concentration is what objectively herein may be said to be a low bacteria concentration.
  • Bacteria without protein and without polysaccharide Bacteria without protein and with polysaccharide (Avicel) Bacteria without protein and with polysaccharide (Avicel)
  • Bacteria with B004_0011 Avicel Bacteria with B004 0010:Avicel
  • sdAb-sdAb fusion protein or “sdAb-sdAb”.
  • compounds of present invention advantageous can remove bacteria at even at low bacteria concentration gives the possibility to intervene early in the course of a bacterial infection (e.g. for prevention and/or prophylaxis related treatment), where the bacteria concentration generally is low.
  • the sdAB-CBM fusion proteins B004_0011 and B004_0010 of Example 5 were bound/loaded onto two different cellulose polymers - Avicel PH-101 (i.e. as in Example 5) and MCC-102 cellulose.
  • MCC-102 may be obtained from provider Sigma Aldrich.
  • MCC-102 Like Avicel pH 101 - MCC-102 is also a cellulose polymer - both are what in the art may be termed microcrystalline cellulose (MCC).

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Abstract

L'invention concerne une composition comprenant une protéine de fusion comprenant un anticorps à domaine unique (sdAb) et un module de liaison aux glucides (CBM) lié à un polymère de polysaccharide par l'intermédiaire d'une liaison non covalente du polymère de polysaccharide au CBM (appelé "sdAb-CBM:pSac") – et son utilisation dans la prévention ou le traitement d'une infection induite par un pathogène dans le tractus gastro-intestinal.
PCT/EP2023/078242 2022-10-14 2023-10-11 Protéine de fusion sdab-cbm liée à un polymère de polysaccharide WO2024079210A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2003031477A1 (fr) * 2001-10-03 2003-04-17 Unilever N.V. Proteines de fusion contenant un domaine de liaison aux hydrates de carbone pour la liberation d'agents therapeutiques et autres agents, et compositions les renfermant
WO2004081026A2 (fr) * 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2020144164A1 (fr) 2019-01-07 2020-07-16 Bactolife Aps Protéines de liaison à des pathogènes

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Publication number Priority date Publication date Assignee Title
WO2003031477A1 (fr) * 2001-10-03 2003-04-17 Unilever N.V. Proteines de fusion contenant un domaine de liaison aux hydrates de carbone pour la liberation d'agents therapeutiques et autres agents, et compositions les renfermant
WO2004081026A2 (fr) * 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2020144164A1 (fr) 2019-01-07 2020-07-16 Bactolife Aps Protéines de liaison à des pathogènes

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