SdAb-CBM fusion proteins bound to polysaccharide polymers

Abstract

A composition comprising a fusion protein (referred to as "sdAb-CBM: pSac") comprising a single domain antibody (sdAb) and a carbohydrate binding module (CBM) bound to a polysaccharide polymer by non-covalent binding of the polysaccharide polymer to the CBM, and its use for the prevention or treatment of infections caused by pathogens in the gastrointestinal tract.

Description

Technical Field

[0001] The present invention relates to a composition comprising a fusion protein, said fusion protein (referred to as "sdAb-CBM:pSac") comprising a single domain antibody (sdAb) and a carbohydrate binding module (CBM), said CBM being bound to a polysaccharide polymer by non-covalent binding of the CBM to the polysaccharide polymer, and to the use of said composition in the prevention or treatment of infections caused by pathogens in the gastrointestinal tract. Background of the Invention

[0003] It is known in the art that nanobodies or single domain antibodies (sdAbs) refer to the smallest antigen-binding fragments or single variable domains ("VHHs") derived from naturally occurring heavy chain antibodies.

[0004] Such single domain antibodies can be derived from antibodies produced in Camelidae species such as camels, llamas, dromedaries, alpacas and guanacos. Single domain antibodies can also be produced synthetically, for example by recombinant expression in a suitable production host cell (such as a bacterial, fungal or mammalian host cell).

[0005] The molecular weight of a single domain antibody (sdAb) is generally much smaller than that of a normal antibody consisting of two heavy protein chains and two light chains.

[0006] Nanobodies have been shown to have the same specificity as antibodies and, in some cases, they are more robust. Their smaller size and single domain make these antibodies easier to transform into microbial (such as bacterial) cells for mass production.

[0007] WO2020 / 144164A1 (Bactolife) describes the use of sdAbs for the treatment of infections caused by pathogens in the gastrointestinal tract (GI) of animals (such as pigs, cows, etc.) or human subjects. The use of sdAb monomers themselves and bivalent sdAb-linker-sdAb constructs is described. For example, Example 13 shows that the bivalent sdAb-linker-sdAb construct can be more effective than the corresponding sdAb monomer.

[0008] It is known in the art that infections caused by pathogens can be caused by the pathogens themselves (e.g., bacteria, such as Escherichia coli), or by molecules produced by the pathogens (e.g., toxins) (e.g., toxin B produced by the pathogen Clostridium difficile).

[0009] It is known in the art that a carbohydrate-binding module (CBM) is a small component of several enzymes, which has an independent fold and function as well as specific carbohydrate-binding activity.

[0010] Many different CBMs are known, and the review article by Oliveira et al. (Biotechnology Advances 33(2015)358-369) outlines different known applications of recombinant CBM-fusion technology, such as including the modification of fibers used in the paper and textile industries, affinity purification tools, and the immobilization of recombinant proteins (e.g., immobilization to carbohydrate-based hydrogels).

[0011] As discussed in the Oliveira review article above, initially, CBMs were classified as cellulose-binding domains (CBDs) because the first examples of these protein domain classes bound to crystalline cellulose.

[0012] The article by Yang et al. (Applied and Environmental Microbiology, April 2020 Volume 86 Issue 8 e02938-19, p1-15) describes the use of cellulose-binding domains to improve the production of nanobodies in the bacterial host cell Bacillus subtilis.

[0013] The article by Hussack et al. (Sensors 2009, 9, 5351-53) describes an immobilization platform for sensing pathogens, biomarkers, and environmental pollutants, which includes sdAbs attached to a CBM and linked to a cellulose support.

[0014] The article by Barbosa et al. (Acta Biomaterialia 143(2022)216-232) describes materials based on antibacterial cellulose peptides (such as hydrogels, papers) for applications such as wound dressings, tissue scaffolds, coatings, etc.

[0015] Without being bound by theory, at the filing date of the present patent application, the inventors were not aware of any prior art document that directly and unambiguously described the following:

[0016] - The use of a fusion protein comprising a single-domain antibody (sdAb) and a carbohydrate-binding module (CBM) (referred to as "sdAb-CBM:pSac") for the prevention / treatment of infections caused by pathogens in the gastrointestinal tract of animal / human subjects, wherein the CBM binds to a polysaccharide polymer through non-covalent binding of the CBM to the polysaccharide polymer. Summary of the Invention

[0017] The problem to be solved by the present invention relates to providing a novel medicament / dietary composition / combination which can be advantageously used, for example, to prevent / treat infections caused by pathogens in the gastrointestinal tract (GI) of animals (e.g., pigs, cattle, etc.) or human subjects, for example, to reduce virulence in the GI and thereby stabilize a healthy microbiome.

[0018] As discussed in further detail herein, the approach is based on the inventors' identification of several surprisingly advantageous properties of a composition comprising a compound that is a fusion protein comprising a single domain antibody (sdAb) and a carbohydrate binding module (CBM) (referred to as "sdAb-CBM:pSac"), wherein the CBM is bound to a polysaccharide polymer via non-covalent binding of the polysaccharide polymer to the CBM, and wherein the composition:

[0019] (i) Contains 10 3 Up to 10 20 Individual sdAb-CBM:pSac compounds per gram (g) weight of the composition.

[0020] The “-” in “sdAb-CBM” refers to covalent binding, and those skilled in the art will understand that it is a fusion protein.

[0021] The ":" in "CBM:pSac" refers to non-covalent binding. As known in the art, CBM is bound to a carbohydrate (eg, a polysaccharide polymer) by non-covalent binding.

[0022] This article Figure 1 Illustrative examples of sdAb-CBM:pSac (referred to as sdAb-CBM:polymer in the figure) compounds are shown, compared to bivalent sdAb-linker-sdAb constructs as prior art in WO2020 / 144164A1 (Bactolife).

[0023] exist Figure 1 In a non-limiting illustrative example, the sdAb binds to a toxin molecule produced by a pathogen.

[0024] Some relevant technical results of the working examples of this article are briefly discussed below.

[0025] Embodiment 1:

[0026] The results of this example show the surprisingly high improved sdAb resistance of sdAb-CBM:pSac compounds to gastric and intestinal proteases, which is clearly advantageous for the treatment of infections caused by pathogens in the gastrointestinal tract of animals / humans.

[0027] Embodiment 2:

[0028] The results of this example showed a surprisingly high / strong binding between the sdAb-CBM fusion protein and cellulose at pH 1.2, i.e., the sdAb-CBM:pSac compound can function at this low pH value, i.e., it can function in gastric acid with a pH between 1 and 3.

[0029] As is known in the art, CBMs are small components of several enzymes, i.e., those skilled in the art would initially expect them to function optimally at the optimal activity pH value of the relevant native enzymes.

[0030] In line with this, see page 363 of the review article by Oliveira et al. discussed above: "CBM binds to cellulose in a moderately wide pH range of 3.5 to 9.5", i.e., the sdAb-CBM fusion protein showed a surprisingly high / good binding to cellulose at pH 1.2, which was unexpected to the inventors.

[0031] Example 3:

[0032] The results of this example essentially demonstrated that it is possible to obtain a composition containing the sdAb-CBM:pSac compound with a very low amount of DNA contamination (if measurable) in a relatively inexpensive / rapid manner.

[0033] This can be an advantage for, e.g., dietary compositions, as the regulatory requirements in the European Union (EU) regarding DNA contamination, etc., are becoming increasingly strict, i.e., a relatively inexpensive / rapid method for removing such DNA contamination can be very useful commercially.

[0034] Example 4:

[0035] The results of this example showed that the binding of sdAb-CBM to cellulose does not interfere with the binding of the antigen, which is clearly important for using sdAb-CBM:pSac to treat infections caused by pathogens discussed herein.

[0036] Example 5:

[0037] The results of this example demonstrated that the sdAb-CBM:pSac compound of the present invention has a surprisingly good ability to remove bacteria even at low bacterial concentrations, which clearly has important clinical significance as it provides, e.g., the possibility of intervening at an early stage of a bacterial infection (when the bacterial concentration is usually low) (e.g., for prevention and / or prophylaxis of related treatments).

[0038] Without being bound by theory, it is believed that the results of the working examples herein, for example, make it possible that a composition comprising a large number of individual sdAb-CBM:pSac compounds is suitable for treating infections caused by pathogens in the gastrointestinal tract of animal / human subjects.

[0039] Without being bound by theory, it is believed that the same composition may also be suitable for other binding proteins of potential interest, such as FAB or DARPin.

[0040] Without being bound by theory, an advantage of the sdAb-CBM:pSac compounds discussed herein may be that, compared to, for example, sdAb-linker-sdAb constructs of the prior art, it is capable of binding a greater number of pathogens / toxins as a single compound, which may facilitate the removal of pathogens / toxins present in small amounts in the gastrointestinal tract (see, for example, Example 5 herein).

[0041] As discussed in WO2020 / 144164A1 (Bactolife), crosslinking of pathogens may be important for the removal of pathogens, for example, resulting in a larger structure that can be relatively easily cleared from the gastrointestinal tract.

[0042] Without being bound by theory, an advantage of the sdAb-CBM:pSac compound may be that, for example, less crosslinking of pathogens / toxins may be required for appropriate clearance / removal because the sdAb-CBM:pSac compound itself is relatively large and can thus be relatively easily removed from the gastrointestinal tract, which can be an advantage, for example, when the content of pathogens / toxins is low and it may be statistically difficult to crosslink a sufficient number of pathogens / toxins to obtain a relatively large molecule that can be relatively easily removed from the gastrointestinal tract.

[0043] If crosslinking is relevant to the removal of a specific pathogen / toxin, the sdAb-CBM:pSac compounds discussed herein may contain many sdAb binding sites, which can be very useful for relatively quickly obtaining crosslinking of many pathogens / toxins.

[0044] Accordingly, a first aspect of the present invention relates to a pharmaceutical composition or dietary composition comprising a compound that is a fusion protein (referred to as "sdAb-CBM:pSac") comprising a single domain antibody (sdAb) and a carbohydrate binding module (CBM), wherein the CBM is bound to a polysaccharide polymer by non-covalent binding of the CBM to the polysaccharide polymer, and wherein the composition:

[0045] (i) contains 10 3 to 10 20 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition;

[0046] And wherein said sdAb binds to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

[0047] The term "individual" in the term "individual sdAb-CBM:pSac compound" of the first aspect should be understood in the way that a person skilled in the art understands in this context, that is, it means that there is no significant binding between individual sdAb-CBM:pSac compounds.

[0048] Thus, after the composition of the first aspect is administered enterally (i.e., via the gastrointestinal tract), the individual sdAb-CBM:pSac compounds in the composition can act actively at different sites of the gastric system, thereby removing pathogens / toxins at different sites of the gastrointestinal tract (GI), that is, treatment of pathogen-induced infections at different sites of the gastrointestinal tract is thus obtained.

[0049] Consistent with the above, there is no significant crosslinking between an individual sdAb-CBM:pSac compound and a polymer of another individual sdAb-CBM:pSac compound in the composition.

[0050] According to the art, the term "pathogen" relates to any organism or agent that can cause disease, where the term disease is widely understood to cover, for example, pathogen-induced infections in the gastrointestinal tract of an animal or human subject, that is, the disease is preferably related to pathogen-induced infections in the gastrointestinal tract of an animal or human subject.

[0051] As mentioned above, the review article by Oliveira et al. and the article by Yang et al. discuss different known applications of recombinant CBM fusion technology, including, for example, modification of fibers used in the paper and textile industries, affinity purification tools, and immobilization of recombinant proteins (e.g., immobilization to carbohydrate-based hydrogels).

[0052] As understood by a person skilled in the art, the pharmaceutical / dietary composition of the first aspect is not directly and explicitly described in any of these prior art articles, for example, because the immobilization of CBM-based paper / textiles and hydrogels clearly cannot produce a composition containing a large number of individual sdAb-CBM:pSac compounds as required by the first aspect.

[0053] In addition, a person skilled in the art understands that the immobilization on a CBM / CBD-based purification column has nothing to do with the pharmaceutical composition or dietary composition of the first aspect.

[0054] As is known in the art, CBM can bind to polysaccharide polymers with a relatively high binding efficiency, so sdAb-CBM and polysaccharide polymers can be administered as a drug combination to a target animal or human subject, for example, for separate or sequential medical uses.

[0055] For example, if the sdAb-CBM fusion protein and the polysaccharide polymer are administered as two separate compositions, it is believed that a sufficient amount of the sdAb-CBM protein and the polysaccharide polymer will be able to find each other in the gastrointestinal tract, thereby producing a sufficient number of sdAb-CBM:pSac compounds in vivo, thereby obtaining the relevant prophylaxis / therapy herein for infections caused by pathogens in the gastrointestinal tract of an animal or human subject.

[0056] As will be understood by those skilled in the art, it is preferable to administer the pharmaceutical composition or dietary composition of the first aspect to an animal or human subject.

[0057] Accordingly, a second aspect of the invention relates to a pharmaceutical combination or dietary combination comprising:

[0058] (A): a pharmaceutical composition or dietary composition comprising from 10 3 to 10 20 individual fusion proteins per gram (g) weight of the composition, wherein the fusion protein (referred to as "sdAb-CBM") comprises a single domain antibody (sdAb) and a carbohydrate binding module (CBM), and wherein the sdAb binds to at least one pathogen surface component and / or at least one molecule produced by the pathogen;

[0059] and

[0060] (B): a pharmaceutical composition or dietary composition comprising a polysaccharide polymer, wherein the polysaccharide polymer is capable of binding to the CBM of (A) by non-covalent binding of the polysaccharide polymer to the CBM, thereby forming a single sdAb-CBM:pSac compound;

[0061] for use simultaneously, separately or sequentially to prevent or treat infections caused by pathogens in the gastrointestinal tract of an animal or human subject.

[0062] As will be understood by those skilled in the art in the context of the present disclosure, for the combination therapies discussed herein, it does not matter whether (A) and (B) are administered simultaneously, for example, as a single composition, or sequentially, for example, as two separate compositions. What matters is that when the second compound / agent is administered, an effective amount of the first compound / agent administered is present in the gastrointestinal tract of the animal / human subject.

[0063] Accordingly, the term "combination" in the second aspect herein relates to various combinations of (A) and (B), for example, in a single pharmaceutical / dietary composition or in separate compositions. The order of administration of (A) and (B) is not important.

[0064] The combination of (A) and (B) can be formulated for simultaneous, separate or sequential administration. Preferably, if not administered simultaneously, the compositions (A) and (B) are administered within a time range relatively close to each other.

[0065] As is known in the art, an infection caused by a pathogen can be caused by the pathogen itself (e.g., a bacterium such as Escherichia coli), or by a molecule produced by the pathogen (e.g., a toxin) (e.g., toxin B produced by the pathogen Clostridium difficile).

[0066] Those skilled in the art should understand that the term "preventing or treating an infection caused by a pathogen in the gastrointestinal tract" should be understood in the way those skilled in the art understand it in the context of the present invention, that is, it broadly encompasses any relevant prevention / treatment of an infection caused by a pathogen herein, such as removing, clearing, blocking and / or inactivating the pathogen / toxin, or for example reducing the risk of infection so that the pathogen does not colonize the gastric system, or for example reducing the virulence in the GI to stabilize the healthy microbiome, or for example so-called microbiome engineering, whereby for example the number of unwanted (e.g., due to possible association with a disease) pathogens (e.g., Escherichia coli) is reduced. For example, if Escherichia coli is suspected to be associated with the development of a related disease (e.g., inflammatory bowel disease (IBD) or an unpleasant condition due to an imbalanced microbiome), the number of possibly relevant Escherichia coli can be reduced.

[0067] The embodiments of the present invention are described below only by way of example. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : Schematic diagram of an example of the sdAb-CBM:pSac compound discussed herein, compared with the bivalent sdAb-linker-sdAb construct of WO2020 / 144164A1 (Bactolife) as the prior art.

[0069] Figure 2 : The gel discussed in Example 3 of the work herein. DETAILED DESCRIPTION OF THE INVENTION

[0071] Drug composition or dietary composition

[0072] Those skilled in the art know whether the composition of interest is a pharmaceutical composition or a dietary composition, i.e., it contains pharmaceutically or dietarily acceptable components.

[0073] The pharmaceutical composition or dietary composition can contain different types of sdAb, CBM and / or pSac.

[0074] For example, the composition can contain different types of sdAb, for example binding to different target pathogens / toxins.

[0075] For example, the composition can comprise some individual sdAb-CBM:pSac compounds (where pSac is, for example, a cellulose polymer) and other individual sdAb-CBM:pSac compounds (where pSac is, for example, a starch polymer).

[0076] As is known in the art, pharmaceutical or dietary compositions can be coated, for example, with an enteric coating to prevent dissolution or disintegration in the gastric environment, which may be preferred when the primary target of the composition is the intestine.

[0077] For a pharmaceutical composition, all components (or excipients) of the composition should be pharmaceutically acceptable. "Pharmaceutically acceptable" refers to non-toxic materials that do not significantly reduce 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)).

[0078] The pharmaceutical composition can also comprise one or more additional active ingredients, such as antibiotics, fecal matter transfer and / or monoclonal antibodies.

[0079] The pharmaceutical composition can be, for example, a tablet, gel, suspension, solution, liposome, capsule, powder or encapsulated in a polymer. Preferably, the pharmaceutical composition is a tablet, capsule or powder.

[0080] For a dietary composition, all components (or excipients) of the composition should be dietarily acceptable.

[0081] In one embodiment, the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers and / or nutrients (such as dietary minerals).

[0082] In one embodiment, the dietary composition is a food, feed or beverage, or a food additive, feed additive or beverage additive, or a food ingredient, feed ingredient or beverage ingredient, or a food supplement, feed supplement or beverage supplement.

[0083] The dietary composition can be, for example, granules (preferred when the dietary composition is feed), tablets, gels, suspensions, solutions, liposomes, capsules, beverages, powders, or encapsulated in a polymer. Preferably, the dietary composition is granules (preferred when the dietary composition is feed), tablets, capsules, beverages, or powders.

[0084] When the subject is an animal and the dietary composition is feed, then most preferably, the dietary composition is a granular composition.

[0085] When the subject is a human, then most preferably, the dietary composition is a tablet or powder, which can be used, for example, as a beverage supplement.

[0086] The dietary composition can be, for example, a so-called medical device composition, such as a medical device composition compliant with, for example, the EU (EU) Medical Device Regulation (MDR) 2017 / 745, such as a so-called class II medical device according to, for example, the MDR.

[0087] The dietary composition can be, for example, a so-called medical food composition.

[0088] For commercially relevant uses, it can be preferred that the pharmaceutical composition or dietary composition of the first aspect and / or its embodiments is a composition with a weight of at least 100 g, more preferably at least 200 g, even more preferably at least 500 g, and most preferably at least 1 kg of the composition itself.

[0089] Fusion protein

[0090] Knowing the relevant sequences of the sdAb and CBM of interest, it is routine work for those skilled in the art to obtain a suitable sdAb-CBM fusion protein herein by using, for example, relevant recombinant expression techniques. That is, it is considered unnecessary to discuss this in great detail herein.

[0091] As mentioned above, the "-" in "sdAb-CBM" refers to a covalent bond, and those skilled in the art can understand that it is a fusion protein.

[0092] As is known in the art, having a suitable linker sequence between the sdAb and CBM can be relevant, that is, the covalent binding of sdAb-CBM does not necessarily have to be a direct covalent binding between the sdAb and CBM.

[0093] A preferred linker is a glycine-serine linker with the structure (G x S) n where x can be a number between 1 and 50, preferably between 1 and 25, more preferably between 1 and 10, and most preferably between 2 and 5, and n refers to the number of repetitions of the GxS sequence, where n can be 1 to 50, preferably 1 to 25, more preferably 1 to 10, and most preferably 2 to 5.

[0094] The sdAb-CBM fusion protein may comprise more than one sdAb, for example two or more sdAbs, which may be, for example, different sdAbs that bind to different pathogens / toxins.

[0095] The fusion protein may also comprise other elements of interest, such as polypeptide / protein sequences, which, for example, are capable of facilitating purification.

[0096] The sdAb-CBM fusion proteins discussed in the working examples herein are all embodiments of sdAb-CBM fusion proteins, wherein the sdAb and CBM are bound together by a covalent peptide bond.

[0097] Preferably, the sdAb-CBM fusion protein of the first and / or second aspect and / or its embodiments is an 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 by a covalent peptide bond, for example directly or for example via a suitable amino acid linker sequence between the sdAb and CBM.

[0098] Single domain antibody (sdAb)

[0099] For the person skilled in the art, obtaining a suitable sdAb related to a suitable target pathogen is routine work, i.e. it is considered unnecessary herein to discuss this in great detail.

[0100] As mentioned above and as is known in the art, a nanobody or single-domain antibody (sdAb) refers to the smallest antigen-binding fragment or single variable domain ("VHH") derived from a naturally occurring heavy-chain antibody.

[0101] Such single-domain antibodies may be derived from, for example, sharks or antibodies produced in camelid species (such as camels, llamas, dromedaries, alpacas and guanacos). Single-domain antibodies may also be produced synthetically, for example by recombinant expression in a suitable production host cell (such as bacteria, fungi or mammalian cells).

[0102] A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that are 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 antibody-derived single-domain scaffolds.

[0103] The term single-domain antibody in its broadest sense is not limited to a particular biological source or a particular method of preparation. For example, the single-domain antibodies of the present disclosure can generally be obtained by: (1) isolating the VHH domain of a naturally-occurring heavy-chain antibody; (2) expressing a nucleotide sequence encoding a naturally-occurring VHH domain; (3) "humanizing" a naturally-occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) "camelizing" a naturally-occurring VH domain from any animal species, particularly from a mammalian species such as from a human, or by expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" as described in the art, or by expressing a nucleic acid encoding such a camelized VH domain; (6) preparing a protein, polypeptide or other amino acid sequence known per se using synthetic or semi-synthetic techniques; (7) preparing a nucleic acid encoding a single-domain antibody using nucleic acid synthesis techniques known per se and then expressing the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0104] As is known in the art, sdAbs can comprise relevant complementarity-determining regions, i.e., in a preferred embodiment, the sdAb comprises the following:

[0105] (i): a complementarity-determining region 1 (CDR1) comprising or consisting of a relevant sequence (e.g., which may be referred to as SEQ ID NO: i);

[0106] (ii): a complementarity-determining region 2 (CDR2) comprising or consisting of a relevant sequence (e.g., which may be referred to as SEQ ID NO: ii); and

[0107] (iii): a complementarity-determining region 3 (CDR3) comprising or consisting of a relevant sequence (e.g., which may be referred to as SEQ ID NO: iii).

[0108] Carbohydrate binding module (CBM)

[0109] As noted above and as is known in the art, a carbohydrate-binding module (CBM) is a small component of several enzymes that has an independent fold and function and specific carbohydrate-binding activity.

[0110] There are many different CBMs known, see for example the review article by Oliveira et al. discussed above.

[0111] As discussed in the Oliveira review article above, initially CBMs were classified as cellulose-binding domains (CBDs) because the first examples of these classes of protein domains bound to crystalline cellulose.

[0112] Thus, a person skilled in the art knows or can routinely determine whether a protein of interest contains a CBM, that is, can routinely determine whether a fusion protein of interest is an sdAb-CBM fusion protein.

[0113] The Oliveira review article cited herein describes that CBMs can be classified into different so-called folding families (see, for example, Table 1 of the Oliveira review article), that is, a person skilled in the art can routinely determine whether a CBM of interest belongs to a specific folding family (such as folding family 3) based on, for example, the Oliveira review article cited herein and relevant common general knowledge.

[0114] Preferably, the CBM belongs to any one of folding families 1-3, more preferably, the CBM belongs to any one of folding families 1 or 3, and even more preferably, the CBM belongs to folding family 3.

[0115] As understood by a person skilled in the art, the preferred selection of a specific CBM is related to the preferred target polysaccharide polymer (see below), that is, if the target polysaccharide polymer is a cellulose polymer, then a suitable CBM can bind to the cellulose polymer.

[0116] If the polysaccharide polymer is a cellulose polymer, the preferred CBM can be a type A CBM.

[0117] As discussed in the Oliveira review article cited herein, type A CBMs or surface-binding CBMs have a flat hydrophobic binding surface composed of aromatic residues, that is, a person skilled in the art can routinely determine whether a CBM of interest is a type A CBM based on, for example, the Oliveira review article cited herein and relevant common general knowledge.

[0118] In the working examples herein, CBMs containing SEQ ID NO:3 or SEQ ID NO:4 were used, thus, preferably, the CBM contains SEQ ID NO:3 or SEQ ID NO:4.

[0119] Polysaccharide polymer

[0120] As is known in the art, a polysaccharide (or polycarbohydrate) is a chain-polymerized carbohydrate composed of monosaccharide units linked together by glycosidic bonds.

[0121] The polysaccharide polymer can be linear or branched.

[0122] The polysaccharide polymer can be a homopolysaccharide or a heteropolysaccharide.

[0123] In the art, some monosaccharide monomers can be modified, yet a person skilled in the art would still objectively consider it a polysaccharide polymer. A person skilled in the art can routinely determine whether a polymer of interest is a polysaccharide polymer based on common general knowledge.

[0124] A polysaccharide polymer may not contain monosaccharide structures / units and still be understood as a suitable polysaccharide polymer herein.

[0125] Relevant aspects herein are, for example, "a carbohydrate-binding module (CBM) binds to a polysaccharide polymer through non-covalent binding of the polysaccharide polymer to the CBM", that is, if the polysaccharide polymer does not contain monosaccharide structures / units, then the CBM of course binds to the polysaccharide polymer part of the polymer itself.

[0126] In the working examples herein, for example, a polysaccharide cellulose polymer containing about 100 to 400 glucose monomers was used.

[0127] A person skilled in the art can routinely determine the preferred number of monomers in a polysaccharide polymer of interest, but preferably at least more than 9 monomers.

[0128] Preferably, the polysaccharide polymer contains 10 to 50,000 monomers, more preferably 10 to 10,000 monomers, even more preferably 10 to 8,000 monomers, and most preferably 50 to 1,000 monomers.

[0129] Preferably, 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.

[0130] As is known in the chemical art, a derivative is a compound derived from a similar compound by a chemical reaction.

[0131] As is known in the art, chitosan can be considered a derivative of chitin, and both chitosan and chitin are polysaccharide polymers.

[0132] Therefore, a person skilled in the art knows the meaning of a derivative of a polysaccharide polymer of interest (such as a derivative of a cellulose polymer). A person skilled in the art can routinely determine whether a polysaccharide polymer of interest is a relevant derivative herein, such as a derivative of a cellulose polymer, based on common general knowledge.

[0133] In a preferred embodiment, the polysaccharide polymer is a cellulose polymer, a hemicellulose polymer, or a starch polymer, more preferably a cellulose polymer or a hemicellulose polymer, and even more preferably a cellulose polymer.

[0134] A preferred example of a hemicellulose polymer is an arabinoxylan polymer.

[0135] Preferably, the polysaccharide polymer is a plant-based / plant-derived polysaccharide.

[0136] The polysaccharide polymer (e.g., preferably a cellulose polymer) can be a crystalline or amorphous polymer. Preferably, the polysaccharide polymer (e.g., preferably a cellulose polymer) is an amorphous polymer.

[0137] Microcrystalline cellulose (MCC) polymer was used in the working examples herein.

[0138] Thus, the polysaccharide polymer (e.g., preferably a cellulose polymer) can be a crystalline polymer, e.g., preferably a microcrystalline cellulose (MCC) polymer.

[0139] If the CBM belongs to either fold family 1 or 3 (even more preferably, if the CBM belongs to fold family 3), then the polysaccharide polymer is particularly preferably a crystalline cellulose polymer (preferably MCC).

[0140] sdAb-CBM:pSac compound

[0141] Based on the common general knowledge and technical teachings herein, a person skilled in the art can routinely obtain the sdAb-CBM:pSac compounds of interest related herein.

[0142] For example, after recombinant expression of, e.g., an sdAb-CBM fusion protein, the polysaccharide polymer can be added to a cell lysate (or supernatant) containing the expressed sdAb-CBM fusion protein, and then the sdAb-CBM:pSac compound can be created / obtained by non-covalent binding of the polysaccharide polymer to the CBM.

[0143] The sdAb-CBM:pSac compound can be obtained in a relatively pure form by, for example, simple centrifugation or more sophisticated known purification techniques.

[0144] As discussed above regarding, e.g., the drug / dietary combination of the second aspect, if the sdAb-CBM fusion protein and the polysaccharide polymer are administered as two separate compositions, then a sufficient amount of the sdAb-CBM protein and the polysaccharide polymer will be able to find each other in the gastrointestinal tract to produce a sufficient amount of the sdAb-CBM:pSac compound in vivo, thereby obtaining prevention / treatment of infections caused by pathogens in the gastrointestinal tract of the relevant animal or human subject herein.

[0145] As discussed above, the first aspect in point (i) relates to the composition containing 10 3 to 10 20 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition.

[0146] Preferably, the composition comprises 10 5 to 10 20 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition; more preferably, the composition comprises 10 5 to 10 19 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition; even more preferably, the composition comprises 10 6 to 10 19 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition; and most preferably, the composition comprises 10 10 to 10 18 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition.

[0147] As described above, the compositions of relevant aspects herein can be, for example, suspensions / solutions.

[0148] As is known in the art, the weight of 1 ml of an aqueous solution is close to 1 mg.

[0149] The compositions of relevant aspects herein can comprise different types of sdAb-CBM:pSac compounds, such as different types of sdAbs directed against different types of pathogens.

[0150] For example, the composition of the first aspect can comprise 10 5 sdAb-CBM:pSac compounds per g, where the sdAb is directed against (i.e., binds to) the Escherichia coli bacterial pathogen, and 10 5 different sdAb-CBM:pSac compounds per g, where the sdAb is directed against (i.e., binds to) another bacterial pathogen, e.g., the composition then comprises 2x10 5 sdAb-CBM:pSac compounds per g.

[0151] Preferred sdAb-CBM:pSac compounds can be, for example, those in which the compound comprises more than one sdAb-CBM that binds to a polysaccharide polymer (see, for example, Figure 1 for exemplary instances).

[0152] For the sdAb-CBM:pSac compound, where the compound comprises more than one sdAb-CBM that binds to a polysaccharide polymer, it may be preferred that more than one (i.e., two or more, such as five or more, or for example ten or more) sdAb-CBMs that bind to a polysaccharide polymer comprise different types of sdAbs (e.g., one sdAb-CBM comprises one type of sdAb, while another sdAb-CBM comprises a different type of sdAb), such that the sdAb-CBM:pSac compound will comprise different types of sdAbs, e.g., these sdAbs can bind to different types of pathogens or different epitopes of the same pathogen.

[0153] The sdAb binds to a pathogen or at least one molecule produced by the pathogen

[0154] As discussed above, relevant aspects herein relate to sdAbs that bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

[0155] As discussed above, for the person skilled in the art, obtaining suitable sdAbs related to a suitable target pathogen is routine work, i.e., it is considered unnecessary herein to discuss this in great detail.

[0156] Preferably, the pathogen is an enterotoxigenic pathogen and / or at least one molecule (preferably an enterotoxin) produced by an enterotoxigenic pathogen.

[0157] As is known in the art, an enterotoxin is a toxin released by a pathogen (e.g., a microorganism) that targets the intestine, i.e., an enterotoxigenic pathogen (preferably an enterotoxigenic bacterium or an enterotoxigenic virus) is a pathogen that targets the intestine.

[0158] Preferably, the pathogen is a bacterium, a virus, a fungus, a protozoan, and / or a worm, more preferably, the pathogen is a bacterium or a virus (e.g., rotavirus or norovirus).

[0159] Preferably, the pathogen is Escherichia coli, Vibrio cholera, Salmonella, Campylobacter, Staphylococcus, Listeria, Shigella, Mycoplasma, and / or Clostridium difficile.

[0160] Preferably, the molecule produced by the pathogen is a toxin, an inhibitor, and / or an enzyme, such as β-lactamase, more preferably, the molecule produced by the pathogen is a toxin.

[0161] Preferably, the toxin is toxin B produced by the pathogen Clostridium difficile, see for example EP21211996.0 (Bactolife - not published at the filing date / priority date of the present application, for example not mentioning anything related to CBM).

[0162] In the working examples herein, the sdAb comprising SEQ ID NO:1 was used, which can be a preferred sdAb.

[0163] The sdAb comprising SEQ ID NO:1 binds to the toxin produced by the pathogen Clostridium difficile.

[0164] Drug composition / dietary composition – other substances

[0165] As described above, the results of Example 3 herein essentially demonstrate that it is possible to obtain a composition comprising the sdAb - CBM:pSac compound with a very low amount of DNA contamination (if measurable) in a relatively inexpensive / rapid manner.

[0166] In short, the method of removing DNA can be seen as based on the inventors' discovery that, surprisingly, very little (if measurable) DNA binds to the sdAb - CBM:pSac compound and that these sdAb - CBM:pSac compounds are relatively easy to precipitate in a liquid medium used for production based on recombinant expression, for example.

[0167] This can be an advantage for, for example, dietary compositions, since, for example, the regulatory requirements of the European Union (EU) regarding DNA contamination and the like are becoming increasingly strict, that is, a relatively inexpensive / rapid method for removing such DNA contamination can be very useful commercially.

[0168] Preferably, the composition of the first aspect (preferably, where the composition is a dietary composition) comprises less than 15 ng DNA / gram (g) of the weight of the composition, more preferably, the composition comprises less than 10 ng DNA / gram (g) of the weight of the composition, and most preferably, the composition comprises less than 5 ng DNA / gram (g) of the weight of the composition.

[0169] The European Food Safety Authority (EFSA) requires that DNA cannot be detected by a method with a detection limit of 10 ng or less (DOI: 0.2903 / j.efsa.2021.6851).

[0170] Use in the prevention or treatment of infections caused by pathogens

[0171] As discussed above, the second aspect relates to a pharmaceutical combination or a dietary combination, which comprises:

[0172] (A): comprising 10 3 to 1020 A pharmaceutical or dietary composition comprising from 100 to 101 individual fusion proteins per gram (g) of the composition by weight, wherein said fusion protein comprises a single domain antibody (sdAb) and a carbohydrate binding module (CBM) (referred to as "sdAb-CBM"), and wherein said sdAb binds to at least one pathogen surface component and / or at least one molecule produced by a pathogen;

[0173] and

[0174] (B): A pharmaceutical or dietary composition comprising a polysaccharide polymer, wherein said polysaccharide polymer is capable of binding to the CBM of (A) by non-covalent binding of the polysaccharide polymer to the CBM, thereby forming a single sdAb-CBM:pSac compound;

[0175] For simultaneous, separate or sequential use for preventing or treating an infection caused by a pathogen in the gastrointestinal tract of an animal or human subject.

[0176] Those skilled in the art will understand that if compositions (A) and (B) are, for example, two separate compositions, then the preferred amount of the sdAb-CBM fusion protein in (A) generally depends on the amount of the polysaccharide polymer in (B). If there are, for example, 10 6 polysaccharide polymers in (B), then it is generally preferred that there are at least 10 6 sdAb-CBM fusion proteins in (A).

[0177] Even more preferably, the number of sdAb-CBM fusion proteins in (A) is significantly higher than the number of polysaccharide polymers in (B) to increase the likelihood of obtaining a single sdAb-CBM:pSac compound, wherein said compound comprises more than one sdAb-CBM bound to the polysaccharide polymer (for example, see the illustrative examples in Figure 1 ).

[0178] As a potentially suitable example, the composition (B) comprising the polysaccharide polymer (for example, a food or feed composition) can be mixed, for example, with the composition (A) comprising the "sdAb-CBM" protein before being administered to the animal or human subject of interest.

[0179] Preferably, composition (A) comprises from 102 to 103 individual sdAb-CBMs per gram (g) of the composition by weight, more preferably, said composition comprises from 103 to 104 individual sdAb-CBMs per gram (g) of the composition by weight, even more preferably, said composition comprises from 104 to 105 individual sdAb-CBMs per gram (g) of the composition by weight 5 to 20 per gram (g) of the composition by weight, even more preferably, said composition comprises from 103 to 104 individual sdAb-CBMs per gram (g) of the composition by weight, even more preferably, said composition comprises from 104 to 105 individual sdAb-CBMs per gram (g) of the composition by weight, even more preferably, said composition comprises from 105 to 106 individual sdAb-CBMs per gram (g) of the composition by weight 5 to 19 per gram (g) of the composition by weight, even more preferably, said composition comprises from 104 to 105 individual sdAb-CBMs per gram (g) of the composition by weight, even more preferably, said composition comprises from 105 to 106 individual sdAb-CBMs per gram (g) of the composition by weight, even more preferably, said composition comprises from 106 to 107 individual sdAb-CBMs per gram (g) of the composition by weight 6 to 19individual sdAb-CBM per gram (g) weight of the composition; and most preferably, the composition comprises 10 10 to 10 18 individual sdAb-CBM per gram (g) weight of the composition.

[0180] Preferably, the pharmaceutical combination is a single composition comprising both compound (A) and compound (B), for example most preferably, wherein the second aspect relates to a pharmaceutical composition or a dietary composition of the first aspect and / or its embodiments for preventing or treating an infection caused by a pathogen in the gastrointestinal tract of an animal or a human subject.

[0181] Preferably, the gastrointestinal tract is the intestine, that is, preventing or treating an infection caused by a pathogen in the intestine of an animal or a human subject.

[0182] As discussed above, the prior art (e.g., WO2020 / 144164A1 (Bactolife)) describes the use of sdAb to treat infections caused by pathogens in the gastrointestinal tract (GI) of animals (e.g., pigs, cows, etc.) or human subjects.

[0183] Therefore, for a person skilled in the art, using the composition / combination described herein for the medical use of the second aspect is routine work, that is, it is considered unnecessary to discuss this in great detail herein.

[0184] Preferably, the animal is a pig, a cow, a poultry, a sheep, a goat, a horse, a chicken, a donkey, a mule, a duck, a goose or a turkey, more preferably, the animal is a pig, a cow or a poultry, and most preferably, the animal is a pig.

[0185] Preferably, the composition is administered enterally, for example orally, such as as a food supplement, as a tablet, chewing gum (e.g., gummy bears) or gel, or by gastric intubation.

[0186] As is known in the art, the term "enterally" relates to administration through the gastrointestinal tract.

[0187] Preferably, the prevention or treatment is for reducing the virulence in the GI, thereby stabilizing the healthy microbiome, especially when the composition is a dietary composition.

[0188] Separate aspect of the present invention – method for removing DNA

[0189] As discussed above, the inventors have found that, surprisingly, a small amount of DNA (if measurable) binds to the sdAb-CBM:pSac compound, and it is believed that the same situation may also apply to other proteins (Prot) that may be of interest.

[0190] Accordingly, a separate aspect of the present invention relates to a method of obtaining a composition comprising a compound, which is a fusion protein (referred to as "Prot-CBM:pSac"), comprising a protein (Prot) and a carbohydrate-binding module (CBM), wherein the CBM is bound to a polysaccharide polymer by non-covalent binding of the CBM to the polysaccharide polymer, and wherein the composition is:

[0191] (i): comprising 10 3 to 10 20 individual Prot-CBM:pSac compounds;

[0192] wherein the method comprises the following steps:

[0193] (A): recombinantly expressing 10 3 to 10 20 individual Prot-CBMs in a suitable microbial host cell;

[0194] (B): obtaining a sample of the Prot-CBM compound expressed in (A), wherein a significant amount of the host cells have been removed;

[0195] (C): adding pSac to the sample of (B) to produce a sample of 10 3 to 10 20 individual Prot-CBM:pSac compounds;

[0196] (D): precipitating the Prot-CBM:pSac compound of (C) to obtain a sample containing the liquid and the precipitated Prot-CBM:pSac compound; and

[0197] (E): removing DNA from the sample of (D) by separating the liquid from the precipitated Prot-CBM:pSac compound (e.g., by washing) to obtain a composition comprising individual Prot-CBM:pSac compounds with DNA removed.

[0198] Each step of the method can be considered a conventional step known to those skilled in the art.

[0199] For example, if the Prot-CBM compound recombinantly expressed in step (A) is secreted into the culture medium, then a significant amount of the host cells can be removed, for example, by centrifugation to obtain sample (B).

[0200] If the expressed Prot-CBM compound is not secreted (e.g., if Escherichia coli is used as the host cell), cell lysis is usually required.

[0201] A person skilled in the art can routinely determine how much pSac to preferably add in step (C), which generally depends on the amount of the expressed Prot-CBM compound.

[0202] The precipitation of the Prot-CBM:pSac compound in step (D) can be routinely accomplished by, for example, centrifugation or other suitable conventional techniques.

[0203] Step (E) is a conventional step mainly involving separating the liquid containing DNA from the precipitated Prot-CBM:pSac compound, which, as is known in the art, can be accomplished, for example, by washing (e.g., with water or a suitable aqueous buffer / solution).

[0204] Preferably, the protein (Prot) in the DNA removal method of the separate aspect is a single-domain antibody (sdAb).

[0205] Preferably, so much DNA is removed in step (E) that the obtained composition contains less than 15 ng DNA / gram (g) weight of the composition (e.g., after other optional related steps, such as related concentration steps), more preferably, the composition contains less than 10 ng DNA / gram (g) weight of the composition, and most preferably, the composition contains less than 5 ng DNA / gram (g) weight of the composition.

[0206] The advantage of the method of the separate aspect is that it is not necessary to use DNase and / or column-based purification steps to remove DNA. Therefore, a preferred embodiment of the method of the separate aspect involves a method that does not involve using DNase and / or column-based purification steps.

[0207] Other preferred embodiments of the composition of the first aspect described herein (e.g., preferred CBM, preferred pSac, etc.) are also preferred embodiments of the DNA removal method of the separate aspect. Examples

[0208] The following examples cite relevant references to the materials and methods mentioned below, which are not related to the inventive concept of the present invention per se.

[0209] -EP21211996.0 (Bactolife – not published at the filing date / priority date of the present application)

[0210] -Martin et al (BMC Biotechnol. 2006;6:46)

[0211] - Studier et al (Protein Expression and Purification 2005, Volume 41, Issue 1, pp. 207 - 234)

[0212] - Ghamghami et al (AMB Expr 10, 129 (2020))

[0213] - Harmsen et al (Applied Microbiology and Biotechnology 2007, Volume 77, Issue 1, pp. 13 - 22)

[0214] - Fiil et al. (iScience 25, 104003, April 15, 2022)

[0215] Example 1 : Binding to cellulose enhances resistance to gastric and intestinal proteases

[0216] Materials and methods

[0217] Composition:

[0218]

[0219] SEQ ID NO:1 is identical to SEQ ID NO:27 of EP21211996.0 (Bactolife – not published at the filing date / priority date of this application)

[0220] sdAb: The sdAb in sdAB - CBM fusion proteins B004_0005 and B004_0006 and the two sdAb domains in sdAb - sdAb fusion protein B003_0005 both contain SEQ ID NO:1.

[0221] CBM: The two CBMs used are the CBM domains from Trichoderma reesei cellobiohydrolase I 1cel7a (B004_0005) and Trichoderma reesei cellobiohydrolase II cel6a (B004_0006). See the table above.

[0222] Polysaccharide polymer: Cellulose polymer Avicel pH 101 – overall range from ~100 to 400 glucose monomers – from supplier Sigma Aldrich.

[0223] Protein production and sdAb-CBM:polymer assembly

[0224] The sdAb-CBM and sdAb-sdAb fusion proteins were cloned into the pSANG10-3F vector (Martin, 2006), ensuring the 6xHis-tag and 3xFLAG tag at the C-terminus.

[0225] The proteins were expressed in an Escherichia coli strain in self-inducing medium (Studier, 2005). After lysis and extraction of the periplasmic fraction (Ghamghami, 2020), the proteins were purified using immobilized metal affinity chromatography (IMAC).

[0226] The sdAb-CBM:pSac compound of the composition was assembled by incubating Avicel PH-101 and sdAb-CBM (4 μmol sdAb-CBM / g Avicel PH-101) at room temperature with inversion rotation for 1 hour.

[0227] Protease resistance test

[0228] sdAb-CBM:pSac, sdAb-CBM or sdAb-sdAb was mixed with 35 mM NaCl, 0.07 HCl, 200 U / L pepsin pH 2 (simulated gastric fluid (SGF)) or 50 mM K3PO4, 10 mg / mL trypsin pH 6.8 (simulated intestinal fluid (SIF)) to study the resistance to proteases. The remaining full-length proteins were evaluated by SDS-PAGE to assess the degree of protease resistance.

[0229] Results

[0230] The following table shows the remaining full-length proteins (percentage of the full-length proteins detected before treatment) after treatment with SGF or SIF.

[0231]

[0232]

[0233] After 10 minutes of treatment with SGF, the compound sdAb-CBM: polymer was approximately 2-fold more resistant to pepsin than sdAb-CBM and at least 46-fold more resistant than the sdAb-sdAb fusion protein. More surprisingly, after 2 hours of exposure to the simulated gastric environment, full-length sdAb-CBM remained in the sdAb-CBM: polymer composition, while all sdAb-sdAb was degraded after only 10 minutes, and at least 98% of the sdAb-CBM in the composition without the carbohydrate polymer was degraded after 30 minutes. Additionally, after 10 minutes in SIF, the sdAb-CBM: polymer composition showed approximately 2-fold increased resistance to trypsin. And, similar to treatment with SGF, when in the sdAb-CBM: polymer composition, after 120 minutes of treatment with SIF, detectable levels of sdAb-CBM were present, while when not bound to the carbohydrate, full-length sdAb-CBM was undetectable after 30 minutes. Similarly, very little (if any) sdAb-sdAb was detected after 30 minutes of treatment with SIF and none after 120 minutes.

[0234] The assembly of sdAb-CBM: polymer is crucial for increasing resistance to proteases. This can be seen from the observation that a composition containing sdAb-sdAb and Avicel PH-101 did not show increased resistance to proteases.

[0235] It is well known in the art that sdAbs have high physicochemical stability and resistance to proteases (Harmsen, 2007, Asaadi 2021). Therefore, it was very surprising to the inventors that the compound (sdAb-CBM:pSac) showed increased resistance to SGF and SIF both in the short term (10 minutes) and at extended times (120 minutes).

[0236] Conclusion

[0237] The results of this example show that the sdAb-CBM:pSac compound can surprisingly highly improve the resistance of sdAb to gastric and intestinal proteases, which is of course beneficial for treating infections caused by pathogens in the gastrointestinal tract of animals / humans.

[0238] Example 2 : The sdAb-CBM fusion protein binds to cellulose under high temperature and low pH conditions

[0239] Materials and methods

[0240] The sdAb-CBM sequence and protein expression and cleavage were the same as in Example 1.

[0241] Incubate the periplasmic fraction from lysed cells with Avicel PH-101 (1 g Avicel PH-101 / 100 mL culture) for 1 hour at room temperature with end-to-end rotation to assemble the sdAb-CBM:polymer composition. Wash the sdAb-CBM:polymer with 150 mM NaCl (10 mL / g Avicel PH-101). Treat the sdAb-CBM-polymer with 10 mL HCl (pH 1.2) / g Avicel PH-101. Evaluate the absorption of sdAb-CBM to Avicel PH-101 by SDS-PAGE before and after treatment.

[0242] Results

[0243] After treatment of the sdAb-CBM:polymer with HCl pH 1.2, approximately 98% of the sdAb-CBM remained bound to Avicel pH-101. Based on previous reports on the binding of CBM to cellulose at pH 3.5 to 9 (Oliverira, 2015), such a high degree of binding at a significantly lower pH value must be regarded as surprising.

[0244] Conclusion

[0245] The results of this example show the surprising high / good binding of the sdAb-CBM fusion protein to cellulose at pH 1.2, i.e., the sdAb-CBM:pSac compound can function at this low pH value, i.e., it can function in gastric acid with a pH of 1 to 3.

[0246] Example 3 : Binding to cellulose reduces DNA contamination when expressed in E. coli

[0247] Materials and methods

[0248] The sdAb-CBM sequences, protein expression, and lysis were the same as in Example 1. The periplasmic fraction from the lysed cells was incubated with 80 μL of Ni-NTA beads (Thermo Scientific) / mL cell lysate or 800 μg of Avicel PH-101 (SigmaAldrich) / mL cell lysate for 1 hour at room temperature with end-to-end rotation. The soluble and insoluble (containing sdAb-CBM:avicel or sdAb-CBM:Ni-NTA) fractions were separated by centrifugation. The insoluble fraction was washed 3 times with 1 / 5 the volume of the cell lysate. sdAb-CBM was eluted with 5 M urea and desalted using a PD-10 column (Cytiva). Every 100 μL of cell lysate, avicel eluate, or Ni-NTA eluate was treated with 117 U of DNase I (Applichem Lifescience). The DNA content was evaluated by 1% agarose gel electrophoresis.

[0249] Results

[0250] Figure 2 It was shown that the DNA content in the sdAb-CBM composition expressed in E. coli was significantly reduced by binding to Avicel PH-101 compared to treatment with the nuclease DNase I. Additionally, the DNA content after binding to Avicel PH-101 was similar to that when binding to Ni-NTA beads, which are highly specific for His-tagged proteins.

[0251] Conclusion

[0252] The results of this example essentially demonstrated that a composition containing the sdAb-CBM:pSac compound with a very low amount of DNA contamination (if measurable) can be obtained in a relatively inexpensive / rapid manner.

[0253] Example 4 : The binding of sdAb-CBM to cellulose does not interfere with antigen binding

[0254] Materials and methods

[0255] The sdAb-CBM and sdAb-sdAB sequences and protein production were as described in Example 1. The assembly of sdAb-CB:polymer was as described in Example 1, but with 1.5 μmol of sdAb-CBM per gram of avicel. The antigen (Uniprot ID: P18177.3 residues 1 - 543) was inserted into a modified pSANG10 without a FLAG tag. The expression and purification of the antigen as a protein were as described in Example 1.

[0256] sdAb-CBM: The polymer, sdAb-CBM, or sdAb-sdAB was incubated with 3-fold molar excess (relative to the binding site) of antigen at room temperature for 2 hours in an end-to-end rotation manner. Anti-FLAG beads (insoluble fraction) were added, and the antigen:sdAb-CBM and antigen:sdAb-sdAb:antigen complexes were pulled down by centrifugation. The antigen:sdAb-CBM:polymer (insoluble fraction) complex was pulled down by centrifugation. The bound antigen (in the insoluble fraction) as part of the total antigen was evaluated by SDS-PAGE.

[0257] Results

[0258]

[0259] The above table shows that 98% of the available antigen binds to the sdAb-CBM:polymer composition, compared to 76% for sdAb-CBM that does not bind to the carbohydrate polymer and 82% for sdAb-sdAb. This indicates that the assembly of the sdAb-CBM:polymer composition does not interfere with antigen binding. Also, antigen binding can be increased optimally compared to the sdAb-sdAb composition.

[0260] Conclusion

[0261] The results of this example show that the binding of sdAb-CBM to cellulose does not interfere with antigen binding, which is clearly important for the use of sdAb-CBM:pSac to treat infections caused by pathogens as discussed herein.

[0262] Example 5 : Comparison of "sdAb-CBM:pSac" (the present invention) with "sdAb-sdAb" (the prior art) - removal of Escherichia coli from solution

[0263] As discussed above, WO2020 / 144164A1 (Bactolife) describes a bivalent sdAb-linker-sdAb construct, also referred to herein as "sdAb-sdAb fusion protein" or "sdAb-sdAb".

[0264] In this comparative example, the sdAb-CBM:pSac compound of the present invention was compared with the sdAb-sdAb of the prior art (i.e., the same sdAb).

[0265] Materials and methods

[0266] Composition:

[0267]

[0268] sdAb: The sdAbs in the sdAB-CBM fusion proteins B004_0011 and B004_0010, and the two sdAb domains in the sdAb-sdAb fusion protein B001_0003 all contain SEQ ID NO:5.

[0269] SEQ ID NO:5 is known in the art as an sdAb that binds to Escherichia coli. The content of the article by Fiil et al. (iScience 25, 104003, April 15, 2022) is: "K922 (GenBank accession number: AJ810819)".

[0270] CBM: The same cel6a and cel7a as used in Example 1.

[0271] Polysaccharide polymer: Cellulose polymer Avicel pH 101 - see Example 1.

[0272] Protein production and sdAb-CBM:polymer assembly

[0273] Protein production and sdAb-CBM: Polymer assembly was carried out essentially as in Example 1, with only relatively minor changes based on the common general knowledge of those skilled in the art.

[0274] The sdAb-CBM:pSac (of the present invention) compounds are designated B004_0011:Avicel and B004_0010:Avicel.

[0275] The "prior art" comparative sdAb-sdAb compound is designated B001_0003.

[0276] Test the ability to remove Escherichia coli from solution

[0277] Compared with B001_0003 (prior art), the ability of B004_0011:Avicel and B004_0010:Avicel (of the present invention) to remove Escherichia coli from solution was carried out by incubating antigen-presenting ETEC F4+ bacteria with the sdAb-CBM:polymer or sdAb-sdAb, and then centrifuging and quantifying the remaining bacteria in the solution. Specifically, the antigen-presenting ETEC F4+ bacteria stock solution was adjusted to a concentration of 1000 CFU / ml. Then the bacteria stock solution was mixed with B004_0011:Avicel or B004_0010:Avicel or B001_0003 in a 1:1 ratio and incubated for 1 hour at room temperature with end-to-end rotation.

[0278] The bacteria:sdAb-CBM:polymer or bacteria:sdAb-sdAb:bacteria complexes were pulled down by centrifugation at 50 RCF for 30 seconds. Representative portions of the supernatant were plated on LB agar plates and incubated overnight at 30 °C and quantified as CFU / ml.

[0279] The bacterial concentration used was objectively low in this article.

[0280] Results

[0281] The following table shows the test results of the ability to remove E. coli from the solution.

[0282] Bacteria without protein and without polysaccharide (Avicel)

[0283]

[0284] Bacteria without protein but with polysaccharide (Avicel)

[0285]

[0286]

[0287] Bacteria with B004_0011:Avicel

[0288]

[0289] Bacteria with B004_0010:Avicel

[0290]

[0291] Bacteria with B001_0003 - "prior art" comparative sdAb-sdAb

[0292]

[0293]

[0294] Conclusion

[0295] As discussed above, WO2020 / 144164A1 (Bactolife) describes a bivalent sdAb-linker-sdAb construct, also referred to herein as "sdAb-sdAb fusion protein" or "sdAb-sdAb".

[0296] In this comparative example, the sdAb-CBM:pSac compound of the present invention was compared with the prior art related sdAb-sdAb of the comparison (i.e., the same sdAb).

[0297] The results of this example show that, compared to the sdAb-sdAb (B001_0003) related to the comparative (i.e., identical sdAb) prior art, the sdAb-CBM:pSac compounds (B004_0011:Avicel and B004_0010:Avicel) of the present invention have a surprisingly large increase in the ability to remove Escherichia coli from solution.

[0298] Even at low bacterial concentrations, this beneficial effect of removing bacteria is significant, which is clearly very advantageous / useful, for example, for removing pathogens / toxins present in small amounts in the gastrointestinal tract.

[0299] The fact that the compounds of the present invention can advantageously remove bacteria even at low bacterial concentrations provides the possibility of intervening at the early stage of a bacterial infection where the bacterial concentration is usually low (e.g., for preventing and / or precluding related treatments).

[0300] Example 6 : Loading B004_0011 and B004_0010 onto two different celluloses

[0301] Materials and methods

[0302] 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 described in Example 5) and MCC-102 cellulose.

[0303] MCC-102 is available from the supplier Sigma Aldrich.

[0304] Like Avicel pH 101, MCC-102 is also a cellulose polymer and can both be referred to as microcrystalline cellulose (MCC) in the art.

[0305] Results

[0306] The results (data not shown) demonstrate that both the sdAB-CBM fusion proteins B004_0011 and B004_0010 can bind correctly to Avicel pH 101 and MCC-102, i.e., both can function satisfactorily and are thus examples of suitable polymers herein.

[0307] Conclusion

[0308] The results of this example show that sdAB-CBM fusion proteins can bind correctly to different polysaccharide polymers, such as different cellulose polymers.

[0309] List of references

[0310] 1: WO2020 / 144164A1 (Bactolife)

[0311] 2: Oliveira et al. (Biotechnology Advances 33(2015)358 - 369)

[0312] 3: Yang et al. (Applied and Environmental Microbiology, April 2020 Volume86 Issue 8 e02938 - 19, p1 - 15)

[0313] 4: Hussack et al. (Sensors 2009, 9, 5351 - 53)

[0314] 5: Barbosa et al. (Acta Biomaterialia 143(2022)216 - 232)

Claims

1. A pharmaceutical or dietary composition comprising a compound, wherein the compound is a fusion protein (referred to as "sdAb-CBM:pSac") comprising a single domain antibody (sdAb) and a carbohydrate binding module (CBM), wherein the CBM binds to a polysaccharide polymer by non-covalent binding to the polysaccharide polymer, and wherein the composition is: (i): comprising 10 3 to 10 20 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition; and wherein the sdAb binds to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

2. The pharmaceutical or dietary composition according to claim 1, wherein the composition comprises 10 10 to 10 18 individual sdAb-CBM:pSac compounds per gram (g) of the weight of the composition..

3. The pharmaceutical or dietary composition according to any one of claims 1-2, wherein the composition comprises less than 15 ng of DNA per gram (g) of the weight of the composition.

4. The pharmaceutical or dietary composition according to claim 3, wherein the composition is a dietary composition, and wherein the dietary composition is a food, feed or beverage, or a food additive, feed additive or beverage additive, or a food ingredient, feed ingredient or beverage ingredient, or a food supplement, feed supplement or beverage supplement.

5. A pharmaceutical combination or dietary combination, comprising: (A): a pharmaceutical or dietary composition comprising 10 3 to 10 20 individual fusion proteins per gram (g) of the weight of the composition, wherein the fusion protein (referred to as "sdAb-CBM") comprises a single domain antibody (sdAb) and a carbohydrate binding module (CBM), and wherein the sdAb binds to at least one pathogen surface component and / or at least one molecule produced by a pathogen; and (B): a pharmaceutical or dietary composition comprising a polysaccharide polymer, wherein the polysaccharide polymer is capable of binding to the CBM of (A) by non-covalent binding of the polysaccharide polymer to the CBM, thereby forming a single sdAb-CBM:pSac compound; for use simultaneously, separately or sequentially to prevent or treat an infection caused by a pathogen in the gastrointestinal tract of an animal or human subject.

6. The pharmaceutical combination or dietary combination for use according to claim 5, wherein the combination is the pharmaceutical or dietary composition according to any one of claims 1-4.

7. The pharmaceutical or dietary composition for use according to any one of claims 5 - 6, wherein the composition is administered enterally, preferably orally, and wherein the gastrointestinal tract is the intestine.

8. The pharmaceutical or dietary composition for use according to any one of claims 5 - 7, wherein the subject is an animal, and wherein the animal is a pig, a cow or poultry.

9. The pharmaceutical or dietary composition according to any one of claims 1 - 4 or the pharmaceutical or dietary composition for use according to any one of claims 5 - 8, wherein the CBM belongs to any one of fold families 1 - 3.

10. The pharmaceutical or dietary composition according to claim 9 or the pharmaceutical or dietary composition for use according to claim 9, wherein the CBM belongs to fold family 3.

11. The pharmaceutical or dietary composition according to any one of claims 1 - 4 or any one of claims 9 - 10, or the pharmaceutical or dietary composition for use according to any one of claims 5 - 8 or any one of claims 9 - 10, wherein the polysaccharide polymer comprises 10 to 50,000 monomers, more preferably 10 to 10,000 monomers, even more preferably 10 to 8,000 monomers, and most preferably 50 to 1,000 monomers.

12. The pharmaceutical or dietary composition according to any one of claims 1 - 4 or any one of claims 9 - 11, or the pharmaceutical or dietary composition for use according to any one of claims 5 - 8 or any one of claims 9 - 11, wherein the polysaccharide polymer is a cellulose polymer, a hemicellulose polymer or a starch polymer.

13. The pharmaceutical or dietary composition according to claim 12 or the pharmaceutical or dietary composition for use according to claim 12, wherein the polysaccharide polymer is a cellulose polymer.

14. The pharmaceutical or dietary composition according to claim 13 or the pharmaceutical or dietary composition for use according to claim 13, wherein the CBM is a type A CBM.

15. The pharmaceutical or dietary composition according to claim 13 or the pharmaceutical or dietary composition for use according to claim 13, wherein the CBM comprises SEQ ID NO:3 or SEQ ID NO:4, and wherein the sdAb comprises SEQ ID NO:

1.

16. The pharmaceutical or dietary composition according to any one of claims 1 - 4 or any one of claims 9 - 15, or the pharmaceutical or dietary composition for use according to any one of claims 5 - 8 or any one of claims 9 - 15, wherein the pathogen is an enterotoxigenic bacterium.

17. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or any one of claims 9-15, wherein the pathogen is an enterotoxigenic bacterium.

18. The pharmaceutical composition or dietary composition according to any one of claims 1-4, wherein the composition is a dietary composition, and all components (or excipients) of the composition are dietetically acceptable.

19. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or claim 18, wherein the composition is a dietary composition, and the dietary composition is a food, feed or beverage - or a food additive, feed additive or beverage additive - or a food ingredient, feed ingredient or beverage ingredient - or a food supplement, feed supplement or beverage supplement.

20. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or claim 18, wherein the composition is a dietary composition, and the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers and / or nutrients such as dietary minerals.

21. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or claim 18, wherein the composition is a dietary composition, and the dietary composition is in the form of granules, tablets, gels, suspensions, solutions, liposomes, capsules, beverages, powders or encapsulated in a polymer.

22. The pharmaceutical composition or dietary composition according to claim 21, wherein the dietary composition is granules, tablets, capsules, beverages or powders.

23. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or claim 18, wherein the composition is a dietary composition, and the dietary composition is a medical food composition.

24. The pharmaceutical composition or dietary composition according to any one of claims 1-4, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition is a tablet, capsule or powder.

25. The pharmaceutical composition or dietary composition according to any one of claims 1-4 or any one of claims 9-15, or the pharmaceutical combination or dietary combination for use according to any one of claims 5-8 or any one of claims 9-15, wherein the sdAb-CBM fusion protein is a polypeptide sequence, and the sdAb and CBM are bound together by a covalent peptide bond.

26. The pharmaceutical composition or dietary composition according to claim 25, or the pharmaceutical combination or dietary combination for use according to claim 25, wherein the sdAb and CBM are bound together by a suitable amino acid linker sequence between the sdAb and CBM.

27. The pharmaceutical composition or dietary composition according to any one of claims 25-26, or the pharmaceutical combination or dietary combination for use according to any one of claims 25-26, wherein the sdAb-CBM fusion protein has been recombinantly expressed.

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