Pathogen Binding Protein

Abstract

The present invention relates to proteins, compositions and uses thereof, wherein the protein comprises a first peptide having a first binding specificity, a second peptide having a second binding specificity and a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by the pathogen.

Description

Technical Field

[0001] The present invention relates to proteins, compositions and uses thereof, in particular proteins and compositions. The protein comprises a first peptide having a first binding specificity, a second peptide having a second binding specificity and a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by the pathogen. Therefore, the protein and / or composition can be used to prevent or treat infections caused by pathogens. Background Art

[0002] Harmsen et al. 2006 article (Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy) involves recombinant single-domain antibodies (V H Oral application of clone K609 (H fragment). K609 is directed against Escherichia Coli F4 pili, more specifically the tip-adhesin FaeG, and has been shown to reduce Escherichia coli (E. coli)-induced diarrhea in piglets, but since the orally applied K609 single-domain antibody is degraded by proteolysis in the stomach, it is only effective at high doses. Four clones with increased in vitro stability were obtained by selection and DNA shuffling. These single-domain antibodies differ from each other and from K609 by up to 10 amino acid residues. The most stable clone, K922, retained 41% of its activity after incubation in gastric fluid and 90% in jejunal fluid, and in addition had a higher affinity for FaeG than the original K609 clone.

[0003] Virdi et al. 2013 (Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection) is concerned with oral feeding-based passive immunization against post-weaning infections, such as post-weaning diarrhea caused by enterotoxigenic Escherichia coli (ETEC). +ETEC antibodies were designed by fusing the variable domains of llama heavy chain-only antibodies to the Fc portion of porcine immunoglobulins (IgG or IgA) and expressing them in Arabidopsis thaliana seeds. H H-IgA monomers, dimers and secretory IgA.

[0004] The article by Lo et al. 2014 (The molecular mechanism of Shiga Toxinstx2eNeutralization by single-domain antibody targeting the cell receptor-binding domain) reported the discovery and characterization of a single domain antibody isolated from a llama phage display library that has potent neutralizing ability against Stx2e toxin. Edema disease is a serious disease characterized by neurological disease, hemorrhagic lesions, and frequent fatal outcomes. Swine edema disease is caused by Escherichia coli that produces Shiga toxin Stx2e. The article suggests that neutralizing NbStx2e1 could be used in the future to prevent or treat swine edema disease.

[0005] The article by Moonens et al. 2014 (Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli) involves nanobodies against the lectin domain of F18 fimbrialadhesin FedF. These nanobodies that bind to FedF were obtained from llamas.

[0006] The invention described in patent application WO 2014 / 033313 A1 relates to an antibody capable of protecting against ETEC infection in a passive immunization program. The anti-ETEC single domain antibody is fused to the IgA Fc domain and produced in plant seeds. This patent application also relates to anti-ETEC, which is anti-F4 + ETEC single domain antibodies and anti-F18 + ETEC single domain antibodies. Summary of the invention

[0007] Provided herein are proteins comprising a first peptide having a first binding specificity, a second peptide having a second binding specificity, and a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, such as a toxin.Proteins provided herein can be used to prevent and / or treat pathogen-induced infections.

[0008] In one aspect, the present invention provides an isolated nucleic acid molecule encoding the protein described in the above aspects.

[0009] In another aspect, the present invention provides a vector comprising the nucleic acid molecule described in the above aspect.

[0010] In another aspect, the present invention provides a recombinant host cell comprising the nucleic acid molecule or vector described in the above aspects.

[0011] In one aspect, the present invention provides a pharmaceutical composition comprising the protein, nucleic acid, vector and / or recombinant host cell described in the above aspects and optionally one or more pharmaceutically acceptable excipients.

[0012] In another aspect, the present invention provides the protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described in the above aspects for use as a medicament.

[0013] In one aspect, the present invention provides a protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition as described above for preventing or treating an infection induced by a pathogen associated with an internal surface and / or an external surface.

[0014] In another aspect, the present invention provides the protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described in the above aspects for preventing or treating gastrointestinal infection and / or lung infection.

[0015] In another aspect, the present invention provides a protein, wherein the protein comprises a first peptide and a second peptide, each comprising three complementarity determining regions CDR1, CDR2 and CDR3.

[0016] In one aspect, the present invention provides a method for cross-linking a pathogen, the method comprising administering to a subject a therapeutically effective amount of the protein, composition and / or pharmaceutical composition described in any one of the above aspects.

[0017] In one aspect, the present invention provides a method for preventing biofilm, comprising administering to a subject a therapeutically effective amount of the protein, composition and / or pharmaceutical composition described in any one of the above aspects.

[0018] In one aspect, the present invention provides a method for neutralizing pathogens and / or molecules secreted by pathogens, the method comprising administering to a subject a therapeutically effective amount of the protein, composition and / or pharmaceutical composition described in any one of the above aspects.

[0019] In one aspect, the present invention provides a method for treating or preventing an infection caused by a pathogen, such as a gastrointestinal infection and / or a lung infection, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein, composition and / or pharmaceutical composition as described in any of the above aspects.

[0020] In another aspect, the present invention provides a method for producing the protein described in any one of the above aspects, the method comprising culturing the host cell described in any one of the above aspects under conditions allowing expression of the encoded protein.

[0021] According to one aspect, the present invention provides a protein comprising at least two single domain antibodies, wherein the protein is specific for at least a first virulence factor and a second virulence factor.

[0022] According to another aspect, the present invention provides a composition comprising a protein of the present invention and optionally one or more excipients, diluents and / or binders.

[0023] According to one aspect, the present invention provides a composition comprising a first single domain antibody specific for a first virulence factor and a second single domain antibody specific for a second virulence factor.

[0024] According to one aspect, the present invention provides a method for treating or preventing a medical condition in an animal or human, comprising administering the protein or composition of the present invention.

[0025] According to one aspect, the present invention provides non-therapeutic uses of the protein or composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The results of stability testing under conditions similar to those in the gastrointestinal tract are shown on a standard SDS gel. The product bands are marked by boxes.

[0027] Figure 2 :The stability test results under high protease concentration and low pH conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0028] Figure 3 : The results of stability test under very high protease concentration and low pH conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0029] Figure 4: Binding capacity test results of homodivalent and heterodivalent protein constructs. When the product binds in the ELISA assay, the measured OD450 represents the signal, and the higher the signal means the more product is bound.

[0030] Figure 5 : Results of stability testing of monomeric and homobivalent protein constructs with and without exposure to citric acid. Panel A) shows the amount of unseparated monomeric and homobivalent protein constructs after citric acid shock, i.e., the amount of product remaining in the wells. Panel B) shows the amount of eluted monomeric and homobivalent protein constructs after citric acid shock.

[0031] Figure 6 : Results of flow-induced dispersion analysis (FIDA) of free LT-B and its interaction with LT1 monomer and LT1 homobivalent protein constructs.

[0032] Figure 7 : Panel A) shows the results of thermal stability of the same bivalent protein construct. Panel B) shows the results of pH stability of the same bivalent protein construct. Panel C) shows the results of bile salt stability of the same bivalent protein construct.

[0033] Figure 8 : Panel A) shows the results of stability testing of the same bivalent protein constructs exposed to gastric fluid. For one sample, the pH was adjusted to about 3 with HCl. The samples were incubated at 37°C for 60 minutes or 120 minutes. Panel B) shows the results of stability testing of the same bivalent protein constructs exposed to pig gall over time. Pig gall was mixed with bacteria and BSA to a final bile concentration of 50%, 10%, or 2%.

[0034] Fig. 9 : Panel A) is a schematic diagram and Panel B) shows an image taken using an optical microscope. Panels A) and B) both show F4 ETEC bacteria without any monomer or bivalent protein construct added, F4 ETEC bacteria with F45 monomers, and F4 ETEC bacteria with F45 homobivalent protein constructs linking the F4 ETEC bacteria together.

[0035] Fig.10: The upper part of Figure A) shows an intestinal epithelial cell and its microvilli on the upper surface. The lower part of Figure A) shows an isolated intestinal villous enterocyte from a piglet with F4 ETEC bacteria, as indicated by the arrows in the left figure. The right figure shows that F4 ETEC bacteria cannot bind to the microvilli of the intestinal epithelial cell when the F45::(GGGS)3::F45 homobivalent protein construct is present. Figure B) shows the efficacy of blocking F4 ETEC binding to porcine villous enterocytes. The efficacy was quantified by counting the intestinal epithelial cells that appeared to bind 0, 1 or 2+F4 ETEC cells compared to the relevant control. 1 is included because floating bacteria may be caught under the coverslip when microscopy is performed. The percentage of intestinal epithelial cells that bind 1 or 2+ETEC cells shows the efficacy of the bacteria in infecting the intestinal epithelial cells. The F183::(GGGS)3::F183 homobivalent protein construct was used as a specificity control.

[0036] Fig.11 : The same bivalent protein constructs for F4 + Results of in vivo treatment of post-weaning diarrhea in piglets (N=32). The x-axis shows the number of days after weaning. The y-axis shows the number of piglets with F4 bacteria in feces (%). Four groups were tested: 1) control: without F45::(GGGGS)3::F45 homobivalent protein construct and without F4, 2) control: with F45::(GGGGS)3::F45 homobivalent protein construct but without F4, 3) without F45::(GGGGS)3::F45 homobivalent protein construct but with F4, and 4) with F45::(GGGGS)3::F45 homobivalent protein construct and with F4.

[0037] Fig.12 : The results of the stability test using porcine gastric juice under conditions similar to those of the gastrointestinal tract are shown on a standard SDS gel. The product bands are indicated by boxes.

[0038] All cited references are incorporated by reference.

[0039] The drawings and examples are provided to explain rather than limit the present invention. It will be appreciated by those skilled in the art that the various aspects, embodiments, claims and any clauses of the present invention may be combined.

[0040] Unless otherwise stated, all percentages are weight / weight. Unless otherwise stated, all measurements are carried out under standard conditions (ambient temperature and pressure). Unless otherwise stated, the test conditions are according to European Pharmacopoeia 8.0. DETAILED DESCRIPTION

[0041] The terms used in this specification generally have their ordinary meanings in the art in the context of the present disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners through the description of the compositions and methods of the present disclosure and how to make and use them.

[0042] definition

[0043] As used herein, the use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". In addition, the terms "having", "including", "containing", and "comprising" are interchangeable, and those skilled in the art know that these terms are open-ended terms.

[0044] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0045] The term "subject" as used herein refers to any subject, such as a human or animal subject. Non-limiting examples thereof may be mammalian subjects and / or livestock. An "effective amount" of a medicament refers to an amount effective to achieve the desired therapeutic or preventive result at the necessary dosage and time period.

[0046] As used herein, the term "combination" refers to the use of more than one therapeutic agent. The use of the term "combination" does not limit the order in which the therapeutic agents are administered to a subject. The first therapeutic agent can be administered before, simultaneously with, or after the second therapeutic agent is administered to the subject.

[0047] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to an intervention in an attempt to alter the natural course of the individual being treated in an amount, manner, and / or pattern effective to improve the condition, symptoms, or parameters associated with the disease, or to prevent the development of the disease to a statistically significant degree or to an extent detectable by one skilled in the art. Treatment can improve, cure, or reduce the duration of a disease or condition in a subject.

[0048] As used herein, the term "monomer" or "monomeric protein" refers to a single peptide with a single binding specificity.

[0049] As used herein, the term "divalent protein construct" refers to a protein comprising a first peptide with a first binding specificity and a second peptide with a second binding specificity connected by a linker.

[0050] As used herein, the term "homodivalent protein construct" refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity connected by a linker, wherein the peptides are identical. "Homodivalent protein construct" may also be referred to herein as "bivalent construct". Therefore, the two terms may be used interchangeably herein.

[0051] As used herein, the term "heterodivalent protein construct" refers to a protein comprising a first peptide with a first binding specificity and a second peptide with a second binding specificity connected by a linker. The peptides are different. "Heterodivalent protein construct" may also be expressed as "heterovalent" herein. Therefore, the two terms can be used interchangeably herein.

[0052] As used herein, the term "surface component" refers to any surface component present on the surface of a pathogen, such as any cell surface component present on the surface of a pathogen. Therefore, the cell surface component may be an adhesion factor, but is not limited thereto.

[0053] As used herein, the term "molecule" refers to any molecule produced by a pathogen. The molecule may be a secreted molecule or a molecule attached to a cell or surface. In some embodiments of the invention, the molecule is a toxin, a protease and / or a binding molecule. In some embodiments of the invention, the molecule is an inhibitor, which can bind to a drug component to inhibit the function of the drug component.

[0054] Protein properties

[0055] The present disclosure relates to a protein comprising

[0056] - a first peptide having a first binding specificity;

[0057] - a second peptide having a second binding specificity; and

[0058] - connectors,

[0059] Wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

[0060] The proteins of the present disclosure are typically isolated proteins, ie, they are not localized or provided within a cell, such as within a cell in a living subject.

[0061] In embodiments of the invention, the protein may comprise one or more further peptides that bind to at least one pathogen surface component and / or at least one molecule produced by the pathogen.

[0062] Single-domain antibodies, i.e. monomers, are usually degraded by proteolysis in the stomach. Therefore, only very large amounts of single-domain antibodies are likely to have an effect in the gastrointestinal tract. Surprisingly, the inventors have found that by connecting at least two peptides using a linker as described in the present invention, the protein construct is stable in the gastrointestinal tract. Interestingly, the inventors have also found that the protein is able to bind to pathogen surface components and / or molecules secreted by pathogens. Therefore, the inventors have produced proteins that effectively prevent and / or treat pathogen-induced infections, cross-link pathogens, prevent biofilm formation, and / or neutralize molecules secreted by pathogens.

[0063] Nature of the joint

[0064] The linker described in the present invention refers to a linker that at least promotes dimerization. Therefore, the present invention relates to a protein, wherein the first peptide and the second peptide are connected via a linker.

[0065] In an embodiment of the present invention, the linker is a stable linker and imparts protein stability of the present invention. Therefore, under exposure as described below, the linker is not degraded and is therefore capable of connecting the first peptide and the second peptide of the present invention. Preferably, the protein is stable in the gastrointestinal tract and / or respiratory system of the subject, that is, the linker can at least promote dimerization of the protein in the gastrointestinal tract and / or respiratory system of the subject. In one embodiment, the protein is acid-stable and / or protease-stable. The protein can be stable in the presence of any acid such as bile acid and / or citric acid. The protein is also preferably stable in the presence of any protease such as pepsin and / or trypsin. In one embodiment, the protein is stable in the presence of bile (gall), for example, in 1 hour under 50%, 10% or 2% bile. In another embodiment, the protein is stable in the presence of bile salts (bile salts), for example, in 1 hour under 0.2mM, 1mM and 10mM sodium deoxycholate (NaDeox).

[0066] In one embodiment of the invention, the protein is temperature stable, for example, temperature stable up to 70°C. This indicates that the protein has high stability. In another embodiment, the protein is pH stable. Unexpectedly, the inventors found that at pH 3-4, about 70-90% of the protein remained stable, so the protein was able to pass through the gastrointestinal tract at least as a dimeric protein and maintain its activity.

[0067] According to one embodiment, the present invention relates to a protein, wherein a first peptide and a second peptide are linked together via a linker.

[0068] In some embodiments of the invention, the first peptide and the second peptide are linked by a GS linker.

[0069] A non-limiting example herein is at least two single domain antibodies linked together by a GS linker. The GS linker is a fusion protein linker consisting primarily of stretches of glycine and serine residues.

[0070] According to one embodiment, the present invention relates to a protein, wherein the GS linker is a protein having (G x S) n The structure of the linker, wherein x refers to a plurality of consecutive G, wherein x can be between 1-10, for example, 1, preferably 2, more preferably 3, preferably 4, preferably 5, more preferably 6, preferably 7, preferably 8, more preferably 9, preferably 10. n refers to G xS sequence is repeated times, where n can be between 1-10, for example 1, preferably 2, more preferably 3, preferably 4, preferably 5, more preferably 6, preferably 7, preferably 8, more preferably 9, and preferably 10. Examples include, but are not limited to, a GGGGS linker (SEQ ID NO: 20), a GGGGSGGGGS linker (SEQ ID NO: 21), a GGGGSGGGGSGGGGS linker (SEQ ID NO: 22), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 23), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 24), or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 25).

[0071] In one embodiment, the GS linker is a GGGGSGGGGSGGGGS (SEQ ID NO: 22) linker.

[0072] The inventors have found that after adding proteases such as pepsin and trypsin, a protein comprising two monomers connected by an IgG3 linker is unstable (see Example 6). After adding a protease, the IgG3 linker is degraded and the two monomers are no longer connected.

[0073] According to one embodiment, preferably, the first peptide and the second peptide of the present invention are not connected by an IgG3 linker. Therefore, the first peptide and the second peptide can be connected by any stable linker, provided that the linker is not an IgG3 linker, such as the IgG3 hinge linker of SEQ ID NO: 26.

[0074] Previous studies have linked the variable domains of llama heavy chain-only antibodies to the Fc portion of porcine immunoglobulins (IgG or IgA), a disadvantage of such a construct is that it may result in an unwanted immune response in the treated subject.

[0075] Therefore, according to another embodiment of the present invention, preferably, the linker does not comprise a part of an antibody, such as an Fc region or a part of an Fc region. Therefore, the first peptide and the second peptide can be connected by any stable linker, provided that the linker does not comprise a part of an antibody, such as an Fc region or a part of an Fc region.

[0076] The pathogen surface component may be any component present on the surface of the pathogen. Thus, it may include, but is not limited to, any virulence factor, such as an adhesion factor.

[0077] Properties of peptides

[0078] The first peptide and / or the second peptide may be any peptide having binding specificity for a pathogen surface component and / or a molecule produced by the pathogen.

[0079] The pathogen surface component may be any component present on the surface of the pathogen. Thus, the pathogen surface component may be a pathogen appendage and / or a virulence factor, such as an adhesion factor.

[0080] The molecules described herein may be toxins, inhibitors and / or enzymes.

[0081] Some pathogens are able to produce enzymes to protect themselves, leading to, for example, antimicrobial resistance, such as bacterial antibiotic resistance. These secreted molecules can be proteases or other molecules that can bind to and neutralize antibiotics against antibiotics or inhibitors. Therefore, proteins against such molecules can neutralize pathogen antibiotic resistance.

[0082] In one embodiment, the molecule produced by the pathogen is a beta lactamase.

[0083] Preferably, the first peptide and the second peptide bind to non-pathogen surface components and / or molecules secreted by non-pathogens with very low affinity. A non-limiting example can be that when the protein is provided to a human subject, the protein has low binding specificity to human surface components (non-pathogen surface components).

[0084] In one embodiment of the invention, the first peptide and / or the second peptide is a single domain antibody. In another embodiment, the protein is a DARPin.

[0085] According to one embodiment, the present invention relates to a protein, wherein said protein is homobivalent or heterobivalent.

[0086] The protein of the present invention comprises the first peptide and the second peptide, which can be combined with:

[0087] -same surface composition;

[0088] - different surface compositions;

[0089] -Surface components and molecules;

[0090] - the same molecule; or

[0091] -Different molecules.

[0092] In one embodiment, the surface components are from the same pathogen.

[0093] In one embodiment, the surface components are from different pathogens.

[0094] In one embodiment, the protein of the invention is capable of cross-linking a pathogen.

[0095] In one aspect of the invention, wherein the first peptide and the second peptide bind to the same pathogen surface component, the protein is capable of cross-linking pathogens expressing the same surface component, such as the same pathogen species. In another aspect of the invention, wherein the first peptide and the second peptide bind to the same pathogen surface component, the protein is capable of neutralizing the adhesion of the pathogen and / or fixing the pathogen.

[0096] In one aspect of the invention, wherein the first peptide and the second peptide bind to different pathogen surface components, the protein is capable of cross-linking pathogens expressing different surface components, such as cross-linking different pathogen species, or cross-linking different pathogen surface components present on the same pathogen species. In another aspect of the invention, wherein the first peptide and the second peptide bind to different pathogen surface components, the protein is capable of neutralizing the adhesion of pathogens and / or fixing pathogens.

[0097] In one embodiment, the protein is capable of cross-linking F4+ ETEC bacteria.

[0098] In another embodiment, the protein of the present invention is capable of neutralizing pathogens and / or molecules produced by pathogens.

[0099] In one aspect of the invention, wherein the first peptide and the second peptide bind to pathogen surface components and molecules secreted by the pathogen, the protein is capable of cross-linking molecules with the pathogen, such as neutralizing toxins secreted by the pathogen.

[0100] In one aspect of the invention, wherein said first peptide and said second peptide are bound to the same molecule, said protein is capable of neutralizing the molecule, for example neutralizing the effect of a secreted toxin and / or a secreted protease.

[0101] In one aspect of the invention, wherein said first peptide and said second peptide are bound to different molecules, said protein is capable of neutralizing at least two different molecules, for example neutralizing the effect of a secreted toxin and / or a secreted protease (possibly in a combined manner).

[0102] In another embodiment, the protein of the present invention is capable of preventing biofilm formation.

[0103] In one embodiment, the dimeric form of the protein has increased binding affinity compared to the monomeric protein.For example, the binding affinity of a homodimeric protein construct comprising two identical single domain antibodies is at least 1.5 times higher than that of the monomeric single domain antibody alone.

[0104] In one embodiment, one of the first peptide or the second peptide binds to an Escherichia coli bacterium, such as F4 + Escherichia coli bacteria and / or F18 + Escherichia coli bacterial binding.

[0105] In one embodiment, one of the first peptide or the second peptide is conjugated to F4 + Escherichia coli bacteria bind, and another peptide binds F18 + Escherichia coli bacteria.

[0106] In one embodiment, the surface component is selected from F18 + 、F4 + , Stx2e, LT and their animal and human variants.

[0107] According to one embodiment, the present invention relates to a protein comprising at least two single domain antibodies, wherein said protein is specific for at least a first virulence factor and a second virulence factor.

[0108] Virulence factors are molecules produced by and / or as part of a microorganism and that cause disease.

[0109] Single domain antibodies are also called “V H H fragment". Single domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. Like a complete antibody, it can selectively bind to a specific antigen. The molecular weight of a single domain antibody is only 12-15kDa, much smaller than the common immunoglobulin G antibody (150-160kDa) composed of two protein heavy chains and two light chains. Single domain antibodies are even smaller than Fab fragments (~50kDa, one light chain and half a heavy chain) and single chain variable fragments (~25kDa, two variable domains, one from the light chain and one from the heavy chain). The first single domain antibody was engineered from the heavy chain antibodies found in the camelid family. These are called V H H fragment.

[0110] V H H may be derived from a camelid heavy chain antibody and is naturally free of light chains.

[0111] The term "single domain antibody construct" refers to at least one single domain antibody connected to at least one other single domain antibody or peptide via a linker.

[0112] The term "nanobody" refers to single domain antibodies derived from natural sources such as camel, llama or shark, which have been selected and optimized for further use.

[0113] According to one embodiment, the present invention relates to a protein, wherein said second virulence factor is the same as or different from said first virulence factor.

[0114] According to one embodiment, the present invention relates to a protein, wherein said protein comprises a first single domain antibody specific for said first virulence factor and a second single domain antibody specific for said second virulence factor.

[0115] "Specific for..." generally means that the single domain antibody binds to the epitope, and the binding requires some complementarity. According to this definition, when a single domain antibody binds to an epitope more easily than to a random, unrelated epitope, it is said to "specifically bind" to that epitope. The term "specificity" is used herein to quantify the relative affinity with which a single domain antibody binds to a certain epitope.

[0116] As used herein, a protein "specific for a virulence factor" refers to a protein expressed at 5 x 10 -7 M or smaller, 1x 10 -7 M or smaller, 5x 10 -8 M or smaller, 1x 10 -8 M or smaller, 5x 10 -9 M or smaller, 1x 10 -9 M or smaller, 5x 10 -10 M or smaller, 1x10 -10 M or smaller, 5x 10 -11 M or smaller or 1x 10 -11 M or less K d In some embodiments, the protein is present at 1 x 10 -8 M to 1x 10 -10 M of K d In certain other embodiments, the protein is present at 1-5 x 10 -9 M of K d Combine.

[0117] As used herein, "homobivalent" refers to the presence of two identical peptides, e.g. H H fragment.

[0118] As used herein, "heterobivalent" refers to the presence of two different peptides, e.g. H H fragment.

[0119] According to one embodiment, the present invention relates to a protein, wherein the protein is specific for 1, preferably 2, more preferably 3, preferably 4 pathogen surface components, such as virulence factors and / or molecules secreted by pathogens. The virulence factors can be different groups of virulence factors from an organism or the same group of virulence factors.

[0120] According to one embodiment, the present invention relates to a protein, wherein the protein comprises a peptide specific for a virulence factor, such as a single domain antibody, wherein the virulence factor forms part of or constitutes a target protein, wherein the target protein is from the same protein subfamily. Protein subfamilies can be defined according to the classification system of the Structural Classification of Proteins (SCOP) database.

[0121] According to one embodiment, the invention relates to a protein, wherein at least one of said virulence factors is an adhesion factor.

[0122] As used herein, "adhesion factor" refers to a type of virulence factor that is a cell surface component or appendage of a bacterium that promotes adhesion or binding to other cells or surfaces.

[0123] According to one embodiment, the present invention relates to a protein, wherein said first and second single domain antibodies are specific for virulence factors from the same or from two different microorganisms.

[0124] According to one embodiment, the present invention relates to a protein, wherein said first single domain antibody is specific for an adhesion factor of a microorganism and said second single domain antibody is specific for a soluble virulence factor of a microorganism.

[0125] According to one embodiment, the present invention relates to a protein, wherein the one or more microorganisms are selected from the group consisting of Escherichia coli bacteria, Vibrio cholera and Shigella bacteria.

[0126] According to one embodiment, the present invention relates to a protein, wherein the one or more pathogens are selected from F4+ Escherichia coli bacteria and F18+ Escherichia coli bacteria.

[0127] According to one embodiment, the present invention relates to a protein, wherein the first pathogen is F4 + Escherichia coli bacteria and the second pathogen is F18 + Escherichia coli bacteria.

[0128] Enterotoxigenic Escherichia coli (ETEC), shigatoxigenic Escherichia coli (STEC), and verotoxigenic Escherichia coli (VTEC) possess two key virulence factors; adhesion factors, usually fimbriae, and the production of one or more toxins that cause disease symptoms.

[0129] As used herein, “F4 +"Escherichia coli" refers to enterotoxigenic Escherichia coli strains with F4 pili (F4 + ETEC), which secrete one or more toxins, such as heat-labile toxins (LTs), after colonization.

[0130] As used herein, “F18 + "Escherichia coli" refers to enterotoxigenic Escherichia coli strains with F18 pili (F18 + ETEC) that secrete one or more toxins after colonization, such as heat-labile toxins (LT) and / or heat-stable enterotoxins (ST). + STEC strains producing Shiga toxin Stx2e, F18 + VTEC strains produce Shiga-like toxins.

[0131] According to one embodiment, the present invention relates to a protein, wherein the first pathogen produces a heat-labile toxin (LT toxin).

[0132] According to one embodiment, the invention relates to a protein, wherein the second pathogen produces a Shiga toxin, such as selected from the group consisting of stx2e and porcine and human variants of stx2e.

[0133] According to one embodiment, the present invention relates to a protein, wherein the first and / or second pathogen is an ETEC Escherichia coli with a pilus type or pilus attachment, such as F5, F6, F41 or AIFA, a human-specific variant, such as a colonization factor antigen (CFA) or an E. coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII and CFAIV.

[0134] According to one embodiment, the present invention relates to a protein, wherein the first and / or second pathogen produces a Shiga toxin or a Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f and stx2g.

[0135] According to one embodiment, the present invention relates to a protein, wherein the virulence factor is selected from the group consisting of ETEC fimbriae adhesins such as F4 and F18, heat-labile enterotoxins (LT), and Shiga toxins or Shiga-like toxins such as Stx2e, and porcine and human variants thereof.

[0136] According to one embodiment, the present invention relates to a protein, wherein the virulence factor is selected from the group consisting of: F18 +、F4 + , Stx2e, LT and their porcine and human variants.

[0137] In one embodiment of the present invention, the first peptide and / or the second peptide comprises an amino acid sequence having at least 90%, for example at least 95%, for example at least 98%, for example at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and / or SEQ ID NO:10.

[0138] The present invention also encompasses the use of peptides having a certain degree of sequence identity or sequence homology with the amino acid sequences defined herein or with peptides having specific properties defined herein. The present invention particularly encompasses peptides having a certain degree of sequence identity with SEQ ID NO: 1-10 or homologues thereof. Here, the term "homologue" means an entity having sequence identity with the subject amino acid sequence.

[0139] In some embodiments of the present invention, the first peptide and / or the second peptide are essentially composed of four "framework regions", which are respectively referred to in the art and hereinafter as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; "framework region 4" or "FR4"; these framework regions are separated by three "complementarity determining regions" or "CDRs", which are respectively referred to in the art and hereinafter as "complementarity determining region" or "CDR1"; "complementarity determining region 2" or "CDR2"; "complementarity determining region 3" or "CDR3". Therefore, the overall structure or sequence of the first peptide and / or the second peptide can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The first peptide and / or the second peptide imparts the first peptide and / or the second peptide with the first and / or second binding specificity.

[0140] In one embodiment, the first peptide and / or the second peptide comprises CDR1, CRD2 and / or CDR3 within the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and / or SEQ ID NO:10.

[0141] In one embodiment, any sequence variation is outside the CDRs.

[0142] In one embodiment, the sequence variation is within the framework regions of the first peptide and / or the second peptide.

[0143] In one embodiment, the first peptide and / or the second peptide comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:27, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:29, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:31.

[0144] In one embodiment, the first peptide and / or the second peptide comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:28, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:30, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:32.

[0145] In one embodiment, the protein comprises a first peptide and a second peptide, wherein the first peptide comprises three complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 27, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31, and wherein the second peptide comprises three complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 32. In one aspect, the homologous amino acid sequence should provide a peptide that retains the functional activity of the peptides of SEQ ID NOs: 1-10.

[0146] In one embodiment, the first peptide and / or the second peptide of SEQ ID NOs: 1-4 and SEQ ID NO: 10 binds to FaeG on F4+ Escherichia coli bacteria.

[0147] In another embodiment, the first peptide and / or the second peptide of SEQ ID NO: 5 binds to the B subunit of Stx2e.

[0148] In another embodiment, the first and / or second peptide of SEQ ID NOs: 6-8 binds to FedF on F18+ Escherichia coli bacteria.

[0149] In another embodiment, the first peptide and / or the second peptide of SEQ ID NO: 9 binds to the B subunit of LT.

[0150] In this context, homologous sequences are considered to include amino acid sequences that may be at least 90%, at least 95%, at least 98% or at least 99% identical to the subject sequence. Typically, homologs will contain the same active sites, etc. as the subject amino acid sequence. Although homology can also be considered similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred that homology means sequence identity.

[0151] In one embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide and the second peptide both comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0152] In another embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide and the second peptide both comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0153] In another embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide and the second peptide both comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5.

[0154] In another embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide and the second peptide both comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0155] In another embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10, and wherein the second peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0156] In another embodiment, the protein described in the present invention comprises a first peptide and a second peptide, wherein the first peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9, and wherein the second peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0157] Nucleic Acids

[0158] In one aspect, the invention provides isolated nucleic acid molecules encoding the proteins described herein.

[0159] "Nucleic acid molecules" include DNA (eg, genomic DNA or complementary DNA) and mRNA molecules, which may be single-stranded or double-stranded. "Isolated" means that the nucleic acid molecule is not located or otherwise provided within a cell.

[0160] Carrier

[0161] In one aspect, the invention provides a vector comprising a nucleic acid molecule as described herein. In one embodiment, the vector is an expression vector.

[0162] Host cells

[0163] In one aspect, the invention provides a recombinant host cell comprising a nucleic acid molecule or vector as described herein. In one embodiment, the host cell is a bacterial, fungal, such as a yeast and / or mammalian cell.

[0164] In one embodiment, the bacterium of the present invention may be a Bacillus, such as Bacillus licheniformis, Bacillus subtilis and / or Bacillus lactobacillus.

[0165] In another embodiment, the yeast of the present invention may be any species selected from the genera Pichia, Hansenula and Saccharomyces.

[0166] In another embodiment, the fungus of the present invention may be Aspergillus oryzae and Aspergillus niger.

[0167] Composition

[0168] Pharmaceutical composition

[0169] The present invention also provides a pharmaceutical composition comprising a protein, nucleic acid, vector and / or recombinant host cell as described herein and further comprising one or more compounds. The compound may be an active compound and / or an excipient.

[0170] In one embodiment, the active compound and / or excipient is one or more complex carbohydrates, such as cellulose and / or alginate, enzymes, activated carbon, surfactants, antibiotics, antiadherents, binders, coatings, disintegrants, fillers, solvents / cosolvents, flavorings, pigments, lubricants, glidants, preservatives, adsorbents, sweeteners, carriers, polymers, buffers, antioxidants, wetting agents, defoamers, thickeners, humectants, and mixtures thereof.

[0171] According to one embodiment, the present invention relates to a composition comprising a protein and optionally one or more excipients, diluents, proteins and / or binders. According to one embodiment, the composition of the present invention may further or optionally comprise one or more of the following: an adhiron, an adnectin, an affibody, an affitin, anticalin, an armadillo repeat protein, an avimer, a beta-hairpin mimetics, a bicyclic peptide, a DARPin and / or a fynomer.

[0172] The present invention also provides the above-mentioned proteins, and compositions comprising one or more of the above-mentioned proteins, and / or pharmaceutical compositions comprising one or more of the above-mentioned proteins.

[0173] According to one embodiment, the present invention relates to a composition comprising a first single domain antibody specific for a first virulence factor and a second single domain antibody specific for a second virulence factor.

[0174] According to one embodiment, the present invention relates to a composition, wherein the second single domain antibody is the same as or different from the first single domain antibody.

[0175] According to one embodiment, the invention relates to a composition, wherein said second virulence factor is the same as or different from said first virulence factor.

[0176] According to one embodiment, the present invention relates to a composition comprising single domain antibodies specific for 2, preferably 3, more preferably 4, preferably 5, more preferably 6, preferably 7, more preferably 8, preferably 9, more preferably 10, preferably 11, more preferably 12 different virulence factors. According to one embodiment, the single domain antibody may form part of one or more, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 proteins.

[0177] According to one embodiment, the present invention relates to a composition comprising a protein of the present invention, and one or more additional single domain antibodies, preferably 1-5, more preferably 2-3 different single domain antibodies, wherein each of the different single domain antibodies-domain antibodies is specific for at least one virulence factor.

[0178] According to one embodiment, the present invention relates to a protein or a composition, wherein the protein or the composition comprises a first single domain antibody specific for the first virulence factor and a second single domain antibody specific for the second virulence factor.

[0179] According to one embodiment, the invention relates to a composition, wherein at least one of said virulence factors is an adhesion factor.

[0180] As used herein, "adhesion factor" refers to a virulence factor that is a cell surface component or appendage of a bacterium that promotes adhesion or binding to other cells or surfaces.

[0181] According to one embodiment, the present invention relates to a composition, wherein said first and second single domain antibodies are specific for virulence factors from the same or from two different microorganisms.

[0182] According to one embodiment, the present invention relates to a composition, wherein the first single domain antibody is specific for an adhesion factor of a microorganism and the second single domain antibody is specific for a soluble virulence factor of a microorganism.

[0183] According to one embodiment, the present invention relates to a composition, wherein the one or more microorganisms are selected from the group consisting of Escherichia coli bacteria, Vibrio cholera and Shigella bacteria.

[0184] According to one embodiment, the invention relates to a composition, wherein the virulence factor is selected from the group consisting of ETEC fimbriae adhesins such as F4 and F18, heat-labile enterotoxins (LT), and Shiga toxins or Shiga-like toxins such as Stx2e, and porcine and human variants thereof.

[0185] According to one embodiment, the present invention relates to a composition, wherein the virulence factor is selected from the group consisting of: F18 + 、F4 + , Stx2e, LT and their porcine and human variants.

[0186] disease

[0187] In one aspect, the present invention provides a protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition as described herein for use as a medicament.

[0188] In another aspect, the invention provides proteins, nucleic acids, vectors, recombinant host cells or pharmaceutical compositions as described herein for preventing or treating an infection induced by a pathogen associated with an inner surface and / or outer surface of a subject. In one embodiment, the infection is a gastrointestinal infection. In another embodiment, the infection is a pulmonary infection.

[0189] In one aspect, the protein, nucleic acid, vector, host cell or pharmaceutical composition is administered orally, pulmonary and / or topically. In one embodiment, the administration is by a nebulizer.

[0190] In one embodiment, the protein, nucleic acid, vector, host cell, or pharmaceutical composition is administered to the subject one or more times.

[0191] In one aspect, the invention provides a protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition as described herein for reducing the time span of active infection in a subject. In one embodiment, the time span of active infection is reduced by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0192] In one embodiment, the protein is capable of reducing the time span of active infection of a F4 ETEC infection in a subject, such as a piglet.

[0193] In another aspect, the present invention provides a protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described herein for use in reducing virulence in a subject.

[0194] In one embodiment, the subject is a human. In another embodiment, the subject is a livestock animal, such as an animal selected from the group consisting of pigs, cattle, sheep, goats, horses, chickens, donkeys, mules, ducks, geese and turkeys. Preferably, the subject is a pig.

[0195] In another aspect, the present invention provides a protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition as described herein for use in preventing or treating post-weaning diarrhea (PWD).

[0196] In another aspect, the present invention provides a protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition as described herein for use in preventing or treating edema.

[0197] According to one embodiment, the invention relates to a protein or a composition for use as a medicament.

[0198] According to one embodiment, the invention relates to a protein or a composition for use in treating an animal or a human.

[0199] According to one embodiment, the invention relates to a protein or composition for prophylactic use in animals or humans.

[0200] According to one embodiment, the invention relates to a protein or a composition for non-systemic use and / or wherein the protein does not interact with the stomach lining, and it preferably does not interact with epithelial cells.

[0201] According to one embodiment, the invention relates to a protein or composition, wherein the protein is stable in the gastrointestinal tract.

[0202] “Stable in the gastrointestinal tract” means that a product containing the single domain antibody retains 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of its binding activity after passing through the gastrointestinal tract.

[0203] According to one embodiment, the present invention relates to a protein or a composition, wherein the protein or the composition is used as an additive to an infant formula, is to be used in combination with an infant formula, or is formulated as a part of an infant formula for animals or humans.

[0204] According to one embodiment, the present invention relates to a protein or a composition, wherein the protein or the composition is used as a component of artificial colostrum.

[0205] According to one embodiment, the invention relates to a protein or composition for oral or pulmonary administration.

[0206] According to one embodiment, the present invention relates to a method for treating or preventing a medical condition in an animal or a human, comprising administering the protein or composition.

[0207] According to one embodiment, the present invention relates to the use of the protein or composition in the treatment or prevention of post-weaning diarrhea (PWD).

[0208] In the livestock industry, enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC) are important pathogens causing high mortality and severe production losses. + ETEC, VTEC, and STEC strains with pili are associated with post-weaning diarrhea. After an initial adhesion step facilitated by F4 or F18 pili, ETEC strains produce and secrete LT and / or Stx2e, which stimulate the secretion of electrolytes and water. This leads to dehydration of the intestinal epithelium and watery diarrhea. + STEC strains produce Shiga toxin Stx2e, and F18 + VTEC strains produce Shiga-like toxins. Damage to the vascular endothelium ultimately leads to edema, hemorrhage, and microthrombosis, and is fatal in 90% of all VTEC-infected animals.

[0209] Use of a protein or composition as claimed in any one of the preceding claims for treating or preventing edema disease. Edema disease is a serious disease characterized by neurological disease, hemorrhagic lesions and frequent fatal outcomes. Swine edema disease is caused by enterotoxigenic and verotoxin-producing Stx2e-producing Escherichia coli.

[0210] According to one embodiment, the present invention relates to non-therapeutic uses of the protein or composition.

[0211] According to one embodiment, the present invention relates to non-therapeutic use of a protein or composition to promote growth in livestock.

[0212] According to one embodiment, the present invention relates to the use of a protein or a composition as a mammalian feed or feed additive.

[0213] According to one embodiment, the invention relates to the use of a protein or a composition, wherein the protein or the composition is dissolved or dispersed in water used by a mammal, such as drinking water or water administered in combination with feed.

[0214] According to one embodiment, the present invention relates to the use of a protein or composition in combination with at least one antimicrobial agent.

[0215] According to one embodiment, the invention relates to a use, wherein the antimicrobial agent is zinc oxide.

[0216] According to one embodiment, the present invention relates to the use of a protein or a composition for stabilizing the microbiome of a human or an animal, preferably for preparing a medicament for stabilizing the microbiome of a human or an animal.

[0217] According to one embodiment, the present invention relates to the use of a protein or composition as a non-therapeutic supplement for humans or animals.

[0218] According to one embodiment, the present invention relates to the use of a protein or composition as a protective aid against bacterial enteric infections during travel.

[0219] According to one embodiment, the present invention relates to the use of a protein or composition as a protective adjuvant against bacterial enteric infections in immunocompromised subjects, such as the elderly, people with downregulated immunopotency including HIV patients, organ recipients, patients receiving rheumatic treatment, patients receiving cancer treatment and / or patients receiving treatment for bacterial infections (such as pneumonia and sepsis).

[0220] According to one embodiment, the present invention relates to the use of a protein or composition for preventing or treating an infection caused by Vibrio cholerae.

[0221] According to one embodiment, the present invention relates to the medical use of the protein or composition.

[0222] According to one embodiment, the present invention relates to a method for preparing a protein or composition, wherein at least one single domain antibody is expressed by a fungal host.

[0223] According to one embodiment, the present invention relates to a method for preparing a protein or composition, wherein the fungal host is a yeast strain.

[0224] According to one embodiment, the present invention relates to a method for preparing a protein or a composition, wherein the fungal host is selected from the group consisting of Aspergillus, Pichia, Hansenula and Saccharomyces.

[0225] According to one embodiment, the invention relates to the use of a protein or a composition for the preparation of a food ingredient for animal or human food.

[0226] Use of proteins, compositions and / or pharmaceutical compositions

[0227] In one embodiment, the present invention relates to a method of cross-linking a pathogen, the method comprising administering to a subject an effective amount of a protein, composition and / or pharmaceutical composition as described herein.

[0228] In another embodiment, the present invention relates to a method of preventing biofilm formation, the method comprising administering to a subject an effective amount of a protein, composition and / or pharmaceutical composition as described herein.

[0229] In another embodiment, the present invention relates to a method of neutralizing a pathogen and / or a molecule secreted by a pathogen, the method comprising administering to a subject an effective amount of a protein, composition and / or pharmaceutical composition as described herein. The protein, composition and pharmaceutical composition are as described herein.

[0230] The present invention also relates to the use of a protein, nucleic acid, vector or host cell as claimed in any one of the preceding claims for the preparation of a medicament for preventing or treating an infection induced by a pathogen associated with an internal and / or external surface of a subject.

[0231] Treatment

[0232] In one aspect, the present invention provides a method for treating or preventing a pathogen-induced infection, such as a gastrointestinal infection and / or a pulmonary infection, comprising administering to a subject a therapeutically effective amount of a protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described herein.

[0233] An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered. As used herein, the phrase "therapeutically effective amount" may refer to an amount of a protein sufficient or effective to treat (delay or prevent onset, prevent development, inhibit, reduce or reverse) an infection.

[0234] Pathogens

[0235] The pathogen may be any pathogen, such as a bacterium, virus, fungus, protozoa and / or helminth.

[0236] In one embodiment, the pathogen is an Escherichia coli bacterium, a Vibrio cholera bacterium, a Salmonella bacterium, a Campylobacter bacterium, a Staphylococcus bacterium, a Listeria bacterium, a Shigella bacterium, a Mycoplasma bacterium, and / or a Clostridium difficile bacterium.

[0237] In another embodiment, the pathogen is F4 + Escherichia coli bacteria and / or F18 + Escherichia coli bacteria.

[0238] In another embodiment, one of the first peptide or the second peptide is + Escherichia coli bacteria, and another peptide bound to F18 + Escherichia coli bacterial binding.

[0239] In another embodiment, the pathogen produces a heat-labile toxin (LT toxin).

[0240] In another embodiment, the pathogen produces a Shiga toxin, such as selected from stx2e and porcine and human variants of stx2e, or a Shiga-like toxin.

[0241] In one embodiment, the pathogen is an ETEC Escherichia coli with a pilus type or pilus attachment, such as F5, F6, F41 or AIFA, a human-specific variant such as a colonization factor antigen (CFA) or an E. coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII and CFAIV.

[0242] In one embodiment, the pathogen produces a Shiga toxin or a Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f, and stx2g.

[0243] Production method

[0244] The present invention provides a method for producing the protein described in any one of the above aspects, the method comprising culturing the host cell defined herein under conditions allowing expression of the encoded protein.

[0245] Therefore, the present invention relates to a method for producing a protein as described herein, the method comprising:

[0246] a) transfecting the nucleic acid molecule and / or the vector described above into a host cell to obtain the recombinant host cell described above;

[0247] b) culturing the recombinant host cell under appropriate conditions;

[0248] c) collecting and purifying the protein expressed by the recombinant host cell.

[0249] Thus, recombinant host cells can be cultured under conditions that allow expression of the encoded protein.

[0250] item

[0251] 1. A protein comprising at least two single domain antibodies, wherein the protein is specific for at least a first virulence factor and a second virulence factor.

[0252] 2. The protein according to item 1, wherein the second virulence factor is the same as or different from the first virulence factor.

[0253] 3. The protein according to any one of the preceding items, wherein the at least two single domain antibodies are linked together via a linker.

[0254] 4. The protein according to any one of the preceding items, wherein the at least two single domain antibodies are linked together via a GS linker.

[0255] 5. The protein of item 4, wherein the GS linker has (G x S) n Structure, wherein x can be a number between 1 and 10, preferably 2 to 5, and n refers to G x The number of repetitions of the S sequence, where n can be between 1 and 10, preferably 2 to 5.

[0256] 6. The protein of item 4 or item 5, wherein the GS linker is a GGGGSGGGGSGGGGS linker (SEQ ID NO: 22).

[0257] 7. The protein of any of the preceding items, wherein the protein comprises a first single domain antibody specific for the first virulence factor and a second single domain antibody specific for the second virulence factor.

[0258] 8. The protein of any of the preceding items, wherein the protein is homobivalent or heterobivalent.

[0259] 9. The protein according to any of the preceding items, wherein the protein is specific for 1, preferably 2, more preferably 3, preferably 4 virulence factors.

[0260] 10. A protein as described in any of the preceding items, wherein the protein comprises a single domain antibody specific for a virulence factor, wherein the virulence factor forms a part of or constitutes a target protein, wherein the target protein is from the same protein subfamily.

[0261] 11. The protein according to item 10, wherein the protein subfamily is defined according to the SCOP system.

[0262] 12. A composition comprising the protein of any of the preceding items and optionally one or more excipients, diluents, proteins and / or binders.

[0263] 13. A composition as described in item 12, comprising a plurality of different proteins, each of which is as described in any of the preceding items, wherein the number of proteins is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

[0264] 14. The composition of item 13, wherein the proteins are all specific for one or two virulence factors.

[0265] 15. A composition comprising a first single domain antibody specific for a first virulence factor and a second single domain antibody specific for a second virulence factor.

[0266] 16. The composition of item 15, wherein the second single domain antibody is the same as or different from the first single domain antibody.

[0267] 17. The composition of item 15 or 16, wherein the second virulence factor is the same as or different from the first virulence factor.

[0268] 18. The composition according to any one of items 12 to 17, comprising single domain antibodies specific for 2, preferably 3, more preferably 4, preferably 5 different virulence factors.

[0269] 19. A composition as described in any one of items 12 to 18, comprising the protein as described in any one of items 1 to 11, and one or more additional single domain antibodies, preferably 1 to 5, more preferably 2 to 3 different single domain antibodies, wherein the different single domain antibodies are all specific for at least one virulence factor.

[0270] 20. The protein or composition of any of the preceding items, wherein the protein or composition comprises a first single domain antibody specific for the first virulence factor and a second single domain antibody specific for the second virulence factor.

[0271] 21. A protein or composition as described in any of the preceding items, comprising at least one single domain antibody that neutralizes toxins.

[0272] 22. A protein or composition as described in any of the preceding items, wherein at least one of the virulence factors is an adhesion factor.

[0273] 23. A protein or composition as described in any of the preceding items, wherein the first and second single domain antibodies are specific for virulence factors from the same or two different microorganisms.

[0274] 24. A protein or composition as described in any of the preceding items, wherein the first single domain antibody is specific for an adhesion factor of a microorganism, and the second single domain antibody is specific for a soluble virulence factor of the same or a different microorganism.

[0275] 25. The protein or composition of any one of items 23 to 24, wherein the one or more microorganisms are selected from Escherichia coli bacteria, Vibrio cholerae bacteria, and Shigella bacteria.

[0276] 26. A protein as described in any one of items 23 to 25, wherein the one or more microorganisms are selected from F4 + Escherichia coli bacteria and F18 + Escherichia coli bacteria.

[0277] 27. A protein as described in any one of items 23 to 26, wherein the first microorganism is F4 + Escherichia coli bacteria and the second microorganism is F18 + Escherichia coli bacteria.

[0278] 28. The protein of any one of items 23 to 27, wherein the first microorganism produces a heat-labile toxin (LT toxin).

[0279] 29. The protein of any one of items 23 to 28, wherein the second microorganism produces a Shiga toxin, such as selected from stx2e and porcine and human variants of stx2e, or a Shiga-like toxin.

[0280] 30. A protein as described in any one of items 23 to 29, wherein the first and / or second microorganism is an ETEC Escherichia coli with a pilus type or pilus attachment: for example, F5, F6, F41 or AIFA, a human-specific variant such as a colonization factor antigen (CFA) or an Escherichia coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII and CFAIV.

[0281] 31. The protein of any one of items 23 to 30, wherein the first and / or second microorganism produces a Shiga toxin or a Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f and stx2g.

[0282] 32. A protein or composition as described in any of the preceding items, wherein the virulence factor is selected from ETEC fimbriae adhesins such as F4 and F18, heat-labile enterotoxins (LT), and Shiga toxins or Shiga-like toxins such as Stx2e and porcine and human variants thereof.

[0283] 33. A protein or composition as described in any of the preceding items, wherein the virulence factor is selected from F18 + 、F4 + , Stx2e, LT and their porcine and human variants.

[0284] 34. A protein or composition as described in any of the preceding items for use as a medicament.

[0285] 35. The protein or composition according to item 34, for use in treating animals or humans.

[0286] 36. A protein or composition as described in any of the preceding items for prophylactic use in animals or humans.

[0287] 37. A protein or composition as described in any of the preceding items for non-systemic use, and / or wherein the protein does not interact with the gastric mucosa, and preferably does not interact with epithelial cells.

[0288] 38. A protein or composition as described in any of the preceding items, wherein the protein or composition is stable in the gastrointestinal tract.

[0289] 39. A protein or composition as described in any of the preceding items, wherein the protein or composition is used as an additive to an infant formula, used in combination with an infant formula, or formulated as part of an infant formula for animals or humans.

[0290] 40. The protein or composition of any of the preceding items, wherein the protein or composition is used as a component of or with artificial colostrum and / or natural colostrum.

[0291] 41. A protein or composition as described in any of the preceding items for oral or pulmonary administration.

[0292] 42. A method of treating or preventing a medical condition in an animal or human comprising administering a protein or composition as described in any one of the preceding items.

[0293] 43. Use of a protein or composition as described in any of the preceding items in the treatment or prevention of post-weaning diarrhoea (PWD).

[0294] 44. Use of a protein or composition as described in any of the preceding items in the treatment or prevention of edema.

[0295] 45. Non-therapeutic use of a protein or composition as described in any one of items 1 to 33, 36 to 41.

[0296] 46. ​​Therapeutic or non-therapeutic use of a protein or composition as described in any of the preceding items to promote growth in livestock.

[0297] 47. Use of the protein or composition as described in any one of the preceding items as feed or feed additive for mammals.

[0298] 48. Use of a protein or composition as described in any of the preceding items, wherein the protein or composition is dissolved or dispersed in water for mammals, such as drinking water or water administered in combination with feed.

[0299] 49. Use of a protein or composition as described in any of the preceding items in combination with at least one antimicrobial agent.

[0300] 50. The use according to item 49, wherein the antimicrobial agent is zinc oxide.

[0301] 51. Use of a protein or composition as described in any of the preceding items for stabilizing the microbiome of humans or animals, preferably for preparing a medicament for stabilizing the microbiome of humans or animals.

[0302] 52. Use of a protein or composition as described in any of the preceding items as a therapeutic or non-therapeutic supplement for humans or animals.

[0303] 53. Use of a protein or composition as described in any of the preceding items as a protective aid against bacterial enteric infections during travel.

[0304] 54. The use of a protein or composition as described in any one of the preceding items as a protective adjuvant for bacterial enteric infections in people with impaired immunopotency, such as the elderly, people with downregulated immunopotency including HIV patients, organ recipients, patients receiving rheumatism treatment, patients receiving cancer treatment and / or patients receiving medical treatment for bacterial infections (e.g. pneumonia and sepsis).

[0305] 55. Use of a protein or composition as described in any of the preceding items for preventing or treating an infection caused by Vibrio cholerae.

[0306] 56. A protein or composition as described in any one of the preceding items for medical use.

[0307] 57. A method of preparing a protein or composition as described in any of the preceding items, wherein at least one protein comprising a single domain antibody is expressed by a fungal host.

[0308] 58. The method for preparing a protein or composition according to item 57, wherein the fungal host is a yeast strain.

[0309] 59. A method for preparing the protein or composition of items 57 to 58, wherein the fungal host is selected from the group consisting of Aspergillus, Pichia, Hansenula and Saccharomyces.

[0310] 60. Use of a protein or composition as described in any of the preceding items in the preparation of a food ingredient for animal or human food, preferably wherein the protein or composition is used together with a probiotic, a prebiotic and / or a synbiotic.

[0311] Example

[0312] The following examples are intended to illustrate the invention and should in no way be construed as limiting.

[0313] Example 1: Construction of single domain antibody constructs for expression in Aspergillus oryzae

[0314] All single-domain antibodies used in this study were generated and subsequently selected and optimized in published studies; Lo et al. 2014 (The molecular Mechanism of Shiga Toxin stx2e Neutralization by Single-domain Antibody Targeting the Cell Receptor-binding Domain); Moonens et al. 2014 (Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli); Virdi et al. 2013 (Orally fed seeds producing designer IgAsprotect weaned piglets against enterotoxigenic Escherichia coli infection) and Harmsen et al. 2006 (Selection and optimization of proteolytically stable llamasingle-domain antibody fragments for oral immunotherapy). However, what all single-domain antibodies have in common is that they were obtained by immunizing healthy llamas with F4, F18, stx4e or LT antigens, and blood samples were collected from the animals at specific time points after immunization. Blood lymphocytes as the genetic source of single domain antibodies are isolated from blood samples, and total RNA is extracted. According to the standard method of nucleotide PCR, cloning and connection, the nano antibody gene sequence is cloned into a suitable vector and transformed into Escherichia coli to generate a clone library. In order to express single domain antibodies in Aspergillus oryzae, synthetic genes with different single domain antibodies are constructed as described in Christensen et al.1988 (High Level Expression of Recombinant Genes in Aspergillus Oryzae). For divalent single domain antibody constructs, a joint region, joint (GGGGS) 3 or pig IgG3 hinge region are introduced.

[0315] The single domain antibody constructs and specific targets are described in Table 1.

[0316] Table 1: Single domain antibody constructs

[0317]

[0318]

[0319] Example 2: Selection and production of Aspergillus oryzae expressing single domain antibody constructs

[0320] The plasmid was transformed into Aspergillus oryzae, a filamentous fungus that can produce and secrete proteins during fermentation. The transformation was performed as described in Christensen et al. 1988 (High Level Expression of Recombinant Genes in Aspergillus Oryzae). The transformants producing the single domain antibody construct were identified by an additional band of about 15 kD appearing on the SDS gel compared to the supernatant from the untransformed parent strain. The culture was filtered and the resulting supernatant containing the single domain antibody construct was used for further analysis.

[0321] Example 3: In vitro testing of the binding capacity of homobivalent and heterobivalent protein constructs

[0322] Methods and Materials

[0323] To detect protein binding to bacteria, an ELISA assay was designed. Briefly, Nunc MaxiSorp TM Plate F96 was coated overnight (O / N) at 4°C with approximately 5-25 ng / mL of unlabeled F45::(GGGGS)3::F45 homobivalent protein construct (SEQ ID NO: 13) in phosphate buffered saline (PBS) pH 7.4. After blocking with milk or bovine serum albumin (3%), heat-killed F4+ Escherichia coli (0149:F4) at OD600 = 0.2 was added to the milk or BSA and incubated on the plates at room temperature. After washing in PBS and 1 hour in a 56°C water bath, the bacteria were heat-killed. For detection of E. coli, FLAG-tagged protein constructs encoding F45::(GGGGS)3::F45 (SEQ ID NO: 13) or F45::(GGGGS)3::LT1 (SEQ ID NO: 16) were added after washing. Homodivalent FLAG or heterodivalent FLAG protein constructs were detected using anti-FLAG-HRP (A8592) from SIGMA. Substrate solution (1:1 mixture of H2O2 and tetramethylbenzidine) was used for development, and an equal amount of 2M H3PO4 was added to stop the reaction. Optical density was measured at 450nm using a microplate reader.

[0324] result

[0325] When the product binds in the ELISA assay, the measured OD450 represents the signal, with higher signals meaning more product is bound. Thus, binding of the F45::(GGGGS)3::F45 homobivalent protein construct and the F45::(GGGGS)3::LT1 heterobivalent protein construct was confirmed in an ELISA experiment for binding to F4 ETEC bacteria (see Figure 4 ). The included negative control F183 (SEQ ID NO: 8) is specific for FedF but not FaeG.

[0326] in conclusion

[0327] Both homobivalent and heterobivalent protein constructs were active and bound to F4 ETEC bacteria.

[0328] Example 4: In vitro testing of citric acid stability of single domain antibodies and bivalent protein constructs

[0329] Methods and Materials

[0330] Comparison of citric acid stability between monomeric single domain antibodies and homobivalent protein constructs. Unlabeled F45::(GGGGS)3::F45 homobivalent protein constructs were first used to coat the wells of the plate, then bacteria were added to bind, excess bacteria were washed off, and then one of the F45 single domain antibodies (SEQ ID NO: 10) or F45::(GGGGS)3::F45 homobivalent protein constructs (SEQ ID NO: 13) attached to the FLAG tag was added. After washing, 0.2M citric acid was added to the wells. The citric acid eluate was transferred to another ELISA plate, which was also coated with unlabeled F45::(GGGGS)3::F45 homobivalent protein constructs and bound to bacteria, and then the citric acid was neutralized with NaOH and the single domain antibody-FLAG was allowed to bind to the bacteria again. The two plates were then incubated with anti-FLAG and developed (ELISA) as described in Example 3. The OD450 signal shows the amount of bound product.

[0331] A standard curve was added to each plate to ensure that the values ​​from the products were within the linear range of the ELISA assay.

[0332] result

[0333] exist Figure 5In A, the high signal corresponds to a higher binding affinity, which is attributed to the inability of the product to be detached with citric acid. After citric acid shock, the F45::(GGGGS)3::F45 homobivalent protein construct was retained in the wells at a higher amount compared to the F45 single domain antibody. Thus, it was demonstrated that the homobivalent protein construct bound better to the bacteria after citric acid shock than the single domain antibody ( Figure 5 A).

[0334] Figure 5 B shows the quantification of the F45 single domain antibody and F45::(GGGGS)3::F45 homobivalent protein construct eluted after citric acid pulse, and is therefore a measure of how easily the product can be washed away from ETEC bacteria. Compared to the single domain antibody, lower amounts of the homobivalent protein construct were detected in the ELISA assay, demonstrating that less homobivalent protein construct was eluted compared to the single domain antibody.

[0335] in conclusion

[0336] We can conclude that the homobivalent protein construct binds with higher affinity than the single domain antibody (ie monomer) since more product remains and less homobivalent protein construct is eluted after acid pulse.

[0337] Thus, after citric acid exposure, the binding affinity of the bivalent protein construct was at least 1.5-fold higher than that of the monomer.

[0338] Example 5: In vitro testing of the thermal stability, pH stability, bile salt stability, stability over time in porcine gastric fluid, and stability over time in porcine bile of the homodivalent protein construct

[0339] Materials and Methods

[0340] ELISA was performed as described in Example 3.

[0341] A standard curve was added to each plate to ensure that the values ​​from the products were within the linear range of the ELISA assay.

[0342] The relative value shows how much signal is detected and therefore the product is bound after incubation, which means that a high signal indicates a high retention and high stability of the signal.

[0343] Thermal stability

[0344] Thermal stability was assessed based on incubation of the F45::(GGGGS)3::F45 homodivalent protein construct for 1 hour at different temperatures followed by assessment of product binding relative to controls in an ELISA setting (see Figure 7 A).

[0345] We can conclude that the homobivalent protein construct is completely stable up to 70°C, indicating high product stability.

[0346] pH stability

[0347] pH stability was assessed based on incubation of the F45::(GGGGS)3::F45 homobivalent protein construct at different pH values ​​for 1 hour at 37°C and then evaluating product binding relative to controls in an ELISA experiment (see Figure 7 B).

[0348] We can conclude that the bivalent protein construct gradually loses stability with decreasing pH, but retains -70-90% activity between pH 3-4, and that the bivalent protein construct is able to pass through the stomach of a subject, such as an animal, and retain biologically relevant activity.

[0349] Bile salt stability

[0350] Sodium deoxycholate (NaDeox) is a bile salt that is added during the step of bacterial binding to the F45::(GGGGS)3::F45-coated bivalent protein construct in the wells. NaDeox was added at concentrations of 0.2 mM, 1 mM, and 10 mM to assess its effect on bacteria-product binding. After incubation, the plates were washed and processed as described above.

[0351] The effect on product-bacteria binding in bile salts was assessed based on incubation of the F45::(GGGGS)3::F45 construct with bacteria at different concentrations of NaDeox for 1 h in an ELISA experiment, and values ​​are expressed relative to the control (see Figure 7 C).

[0352] We can conclude that the homobivalent protein construct remains completely stable and active in the presence of bile salts (also outside the biologically relevant range) and will therefore be active in the intestine.

[0353] Stability over time in porcine gastric juice

[0354] Gastric juice was extracted from the stomach of euthanized pigs (Yorkshire Landrace weighing approximately 30 kg). Gastric juice was clarified by centrifugation within 1-2 hours after extraction and then kept at -80°C until further use. When the pre-treated unlabeled F45::(GGGGS)3::F45 homobivalent protein construct samples were diluted 1:200 in gastric juice, the pH was measured and the pH of one sample was adjusted to approximately 3 using HCl. The samples were then incubated at 37°C for 1 hour and then further diluted and used to coat ELISA plates. For all treatments, all subsequent steps (ELISA) were processed as described in Example 3.

[0355] Product stability in porcine gastric fluid was assessed based on incubation of the F45::(GGGGS)3::F45 homobivalent protein construct with gastric contents from three different piglets at 37°C for 1 hour and then assessment of product binding relative to control in an ELISA experiment. pH = 4 / 5 and the last sample was adjusted to pH 3 using HCl (see Figure 8 A).

[0356] We can conclude that the homobivalent protein construct remains stable in the gastric fluid of piglets and therefore remains active upon oral administration.

[0357] Stability over time in porcine bile

[0358] Bile was extracted from the gallbladder of pigs (Yorkshire Landrace pigs obtained from a specific pathogen-free herd and euthanized at a weight of approximately 30 kg). Bile was stored on ice / refrigerated until further use. Before use, bile was slightly heated at 37°C to dissolve lipids. High levels of bile precipitate milk, so for these assays, BSA (final concentration of 3%) was used in the buffer and blocking buffer. Bile was mixed with bacteria and BSA to a final bile concentration of 50%, 10%, or 2%. After bile / bacteria incubation, the plates were washed and processed as described above.

[0359] The effect on product-bacteria binding in pig bile was evaluated based on incubation of F45::(GGGGS)3::F45 with bacteria (F4 ETEC) for 1 h at different bile concentrations from 3 individual piglets and then evaluating product binding relative to controls in an ELISA setup (see Figure 8 B).

[0360] We can conclude that the homobivalent protein construct remains stable and active in bile and will therefore remain functional in the intestine.

[0361] Overall conclusion

[0362] We can conclude that the homobivalent protein construct is completely stable in the temperature range relevant to humans and animals. The homobivalent protein construct maintains biologically relevant activity when the pH is reduced, and it remains completely stable and active in the presence of bile (gall), so it will be active in the intestine. The homobivalent protein construct also remains stable in the gastric fluid of piglets and therefore remains active when administered orally.

[0363] Example 6: In vitro testing of protease stability of single domain antibody constructs at specific concentrations and pH

[0364] To ensure the stability of the single-domain antibody constructs during oral administration, a stability test with the addition of proteases was performed. The following monomeric single-domain antibodies were used: F41, F45, F183, and the following bivalent single-domain antibody constructs: F183::(GGGGS)3::F183, F183::IgG3::F183. The latter linker sequence has been previously used by Virdi et al. 2013 (Orallyfed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coliinfection) and described as stable. The single-domain antibody constructs were each incubated at 37°C for 1 hour with:

[0365] Pepsin: an endopeptidase found in the digestive system

[0366] Trypsin: a serine protease found in the digestive system

[0367] Pancreatin: A mixture of amylase, lipase, and protease obtained from porcine pancreas

[0368] Gastric juice from pigs

[0369] When the single domain antibody constructs are administered orally at a later stage, proteases and gastric juices are selected to determine the possible stability in the gastrointestinal tract.

[0370] Stability testing was performed using proteases at different concentrations and pH. Samples containing single domain antibody constructs were analyzed using SDS-page before and after incubation with proteases to assess possible protein degradation. Bovine serum albumin (BSA) was used as a reference for normal protein content.

[0371] The specific protease concentrations and pH used were as follows:

[0372] Stability test under normal activity ( Figure 1 ):

[0373] Pepsin 250 μg / mL (incubation pH 3)

[0374] Trypsin 125 μg / mL

[0375] Pancreatin 100 μg / mL

[0376] Stability test under high activity ( Figure 2 ):

[0377] Pepsin 500 μg / mL (incubation pH 1-2)

[0378] Trypsin 250 μg / mL

[0379] Pancreatin 100 μg / mL

[0380] Stability testing at very high activity ( Figure 3 ):

[0381] Pepsin 50 μg / mL (incubation pH 1-2)

[0382] Trypsin 500 μg / mL

[0383] Pancreatin 150 μg / mL

[0384] In stability testing under conditions similar to gastrointestinal conditions, all individual single domain antibody constructs as well as the same bivalent single domain antibody construct F183::(GGGGS)3::F183 were stable. However, another same bivalent single domain antibody construct F183::IgG3::F183 was degraded by trypsin and pancreatin, and a lighter product was visible on the SDS gel. Therefore, surprisingly, the IgG3 linker was degraded by two proteases, trypsin and pancreatin, while the (GGGGS)3 linker remained completely stable. The same surprising pattern was observed after incubation in pig gastric juice, where the same bivalent construct with the IgG3 linker was cleaved, while the same bivalent construct with the (GGGGS)3 linker remained stable ( Fig.12 ).

[0385] At high and very high concentrations of proteases and at lower pH, a greater amount of the single domain antibody constructs were generally degraded. When comparing the individual single domain antibody constructs, differences in stability were observed: F41 was less degraded by pepsin and more by trypsin and pancreatin, while the opposite was observed for F45 at high concentrations of proteases. In addition, at very high concentrations, F45 was more stable than F41. The stability of F45 and F183 was similar under all conditions.

[0386] Example 7: In vitro testing of bacterial aggregation

[0387] Materials and Methods

[0388] F4 Escherichia coli (0149: F4) O / N cultures were grown O / N in LB at 37°C with shaking at 180 rpm. The cultures were then washed once in PBS and the OD600 was adjusted to 1. 1 mL of bacteria were pelleted and resuspended in 40 μl of approximately 600 ng / mL of labeled or unlabeled F45 single domain antibody or the same bivalent F45::(GGGGS)3::F45 same bivalent protein construct. The samples were allowed to settle for a few minutes and then mounted on a slide. Images were taken on an optical microscope using a 100X oil objective.

[0389] result

[0390] Microscopic images of F4 ETEC bacterial cultures treated with and without F45 monomer or F45::(GGGGS)3::F45 products show how the products affect the bacteria. Although F45 did not affect bacterial aggregation or motility, the bivalent protein structure F45::(GGGGS)3::F45 interlinked F4 ETEC and caused them to aggregate, which can be seen as bacterial clumps under the microscope (see Fig. 9 ).

[0391] in conclusion

[0392] Based on these images, we can conclude that the homobivalent protein constructs are active, bind at both ends, and have a cumulative effect greater than the two individual single domain antibodies they comprise, due to the fact that they will promote aggregate formation and block far more binding sites.

[0393] Example 8: In vitro testing of single domain antibody constructs for binding to ETEC bacteria and competition with cell receptors

[0394] Adhesion assays can be established to test the binding of single domain antibody monomers or constructs and competition with natural receptors. This assay can demonstrate the efficacy of the product in isolating ETEC bacteria from binding to porcine epithelial cells. Porcine Intestinal Porcine Epithelial Cell line-1 (IPEC-1) can be inoculated, cultured and then treated with F18 +ETEC bacterial infection to simulate the binding between cells and bacteria in the intestine. ETEC bacterial cells can be stained with DAPI for visualization and measured using flow cytometry. To quantify bacterial load, unstained bacterial cells can be incubated with porcine cells for assays to measure colony forming units. To explore whether the single domain antibody construct can block the adhesion of ETEC bacterial cells to IPEC-1 cells and thereby compete with cell receptors, adhesion assays can be performed with or without the addition of single domain antibody constructs. Single domain antibody monomers, constructs, mixtures of monomers and / or constructs, or any of the above can be tested after treatment with proteases, low pH, or gastric juice (as described in Example 3). Single domain antibody monomers or constructs and F18 + ETEC bacteria binding, and blocking binding to IPEC-1 cells. Another analysis can also be performed to study the effect of F4 in the presence or absence of single domain antibody monomers or constructs. + Adhesion of ETEC bacteria to the IPEC-J2 cell line expressing F4.

[0395] Measurements are performed to determine one or more of the following effects:

[0396] 1. The use of a mixture of multiple single domain antibody constructs has a potentially higher efficacy in sequestering ETEC from adhering to porcine epithelial cells compared to individual single domain antibody monomers or constructs.

[0397] 2. Bivalent constructs have potentially similar or higher efficacy compared to single domain antibody monomers.

[0398] 3. After treatment with biologically relevant proteases from the digestive tract, low pH or porcine gastric fluid (as described in Example 3), the bivalent constructs have potentially similar or greater stability compared to the single domain antibody monomers, as measured by the activity retained by the constructs in the adhesion assay after treatment.

[0399] Example 9: Cytotoxicity assays to determine the toxicity of LT and stx2e and the effects of toxin-specific single domain antibody constructs to isolate toxin binding

[0400] Cytotoxicity assays can be performed to determine the toxicity of ETEC toxins LT and stx2e, and the ability of single-domain antibody constructs to eliminate this toxicity by toxin sequestration. Cells exposed to cytotoxic compounds can respond in a variety of ways; if the toxin is lethal, the cells may undergo necrosis, apoptosis or autophagy, or if the damage is not too severe, the cells may reduce or stop their growth and proliferation. During in vitro cytotoxicity assays, any of these responses can be measured in the presence of LT and stx2e. Vero cells are a cell line established by kidney epithelial cells extracted from African green monkeys, which can be used in this study. Cells can be exposed to different / increasing concentrations of LT and stx2e, and then the cellular response can be measured to determine the severity of the two toxins. In this study, single-domain antibody monomers, constructs, mixtures of the above, or any of the above can be tested after treatment with products such as proteases, low pH or gastric juice (as described in Example 3). It is expected that the single-domain antibody construct will sequester the toxins and thus inhibit the cytotoxic effects of ETEC toxins LT and stx2e. Cytotoxicity assays can be performed using toxin mixtures obtained from ETEC bacterial supernatants. The effects can be dose-dependent.

[0401] Measurements are performed to determine one or more of the following effects:

[0402] 1. The use of a mixture of multiple single domain antibody constructs has a potentially higher efficacy in sequestering the toxic effects of toxins compared to a single single domain antibody monomer or construct.

[0403] 2. Bivalent constructs have potentially similar or higher efficacy compared to single domain antibody monomers.

[0404] 3. After treatment with biologically relevant proteases from the digestive tract, low pH or porcine gastric fluid (as described in Example 3), the bivalent constructs have potentially similar or greater stability compared to the single domain antibody monomers, as determined by measuring the activity retained by the constructs in a cytotoxicity assay after treatment.

[0405] Example 10: In vitro testing of single domain antibody constructs for binding to ETEC bacteria and competition with cell receptors on porcine intestinal villi

[0406] To show the effect of single domain antibodies on ETEC villus binding, small intestinal villus enterocytes can be isolated from the intestinal mucosa of freshly slaughtered piglets. The villi can be incubated with ETEC bacteria in the presence and absence of single domain antibody constructs, and the number of adherent bacteria will be determined using phase contrast microscopy and immunohistochemistry. Single domain antibody monomers, constructs, mixtures of the above, or any of the above can be tested after treatment with products such as proteases, low pH, or gastric juice (as described in Example 3). Adding single domain antibody monomers or constructs prior to incubation with ETEC bacteria can isolate ETEC bacteria from adhering and will show the efficacy of the product.

[0407] Measurements are performed to determine one or more of the following effects:

[0408] 1. Using a mixture of multiple single domain antibody constructs has a potentially higher efficacy in isolating adhesion to porcine intestinal villi compared to individual single domain antibody monomers or constructs.

[0409] 2. Bivalent constructs have potentially similar or higher efficacy compared to single domain antibody monomers.

[0410] 3. The bivalent constructs have potentially similar or greater stability compared to single domain antibody monomers after treatment with biologically relevant proteases from the digestive tract, low pH, or gastric fluid from pigs (see Example 3), as measured by the activity of the constructs retained after treatment in terms of their ability to sequester ETEC bacteria to adhere to pig intestinal villi.

[0411] Example 11: In vitro effects of the homodivalent protein construct on ETEC binding to porcine villus intestinal epithelial cells

[0412] Materials and Methods

[0413] Intestinal villus enterocytes were isolated from piglets and subsequently subjected to adhesion assays as described by Van den Broeck et al. (1999). Briefly, 15-20 cm long intestinal segments were cut from the mid-jejunum of pigs (Yorkshire Landrace pigs obtained from a specific pathogen-free herd and euthanized at 3-4 months of age). The segments were opened and washed in Krebs-Henseleit buffer (120 mM NaCl, 14 mM KCl, 25 mM NaHCO3, 1 mM KH2PO4 [pH 7.4]) and then kept in Krebs Henseleit buffer containing 1% formaldehyde for about 1 hour. Subsequently, the buffer was replaced with sterile PBS (pH 7.4), the villi were gently scraped from the mucosa with a slide, and suspended and washed in sterile PBS (pH 7.4) buffer until the supernatant was clear. The samples were stored in PBS and kept at 4°C until further use.

[0414] F4+ Escherichia coli (0149:F4) were inoculated from a single colony and cultured overnight in LB at 37°C with shaking. The bacteria were washed once in PBS (pH 7.4) and pretreated with a solution containing: unlabeled or labeled F45::(GGGS)3::F45 homodivalent protein construct, F183::(GGGS)3::F183 homodivalent protein construct. The bacteria / F45::(GGGS)3::F45 mixture or bacteria / F183::(GGGS)3::F183 mixture was added to the slurry of villus material and gently rotated at room temperature to allow adhesion to occur. After one hour, the samples were mounted on a slide and images were taken on an optical microscope using a 100X oil objective and quantified manually based on the images taken with the microscope.

[0415] result

[0416] The efficacy of the product in blocking the binding of F4 ETEC to porcine villus intestinal epithelial cells was quantified by counting the number of intestinal epithelial cells that had 0, 1, or 2+F4ETEC cells bound compared to the relevant control. 1 was included because floating bacteria could be caught under the coverslip when performing microscopy. The percentage of intestinal epithelial cells that had 1 or 2+ETEC cells bound indicated the efficacy of the bacteria in infecting the intestinal epithelial cells. F4 ETEC+F183::(GGGS)3::F183 was the control. See Fig.10 .

[0417] in conclusion

[0418] It was concluded that the adhesion of F4 ETEC to intestinal epithelial cells was almost completely stopped with the addition of the F45::(GGGS)3::F45 homobivalent protein construct, which was attributed to the blocking of bacterial binding to intestinal epithelial cells by the homobivalent protein construct.

[0419] Example 12: FIDA was performed to determine the binding of single domain antibodies and their bivalent constructs

[0420] To determine the binding and complex formation between single domain antibodies and their bivalent single domain antibody constructs and analytes, flow induced dispersion analysis (FIDA) can be performed as described in Poulsen et al. 2015 (Flow Induced Dispersion Analysis Rapidly Quantifies Proteins in Human Plasma Samples). All mentioned single domain antibodies and their bivalent single domain antibody constructs can be analyzed to record and quantify analyte binding, including K d And complex formation. Single domain antibody monomers, constructs, mixtures of the above, or any of the above can be tested after treatment with proteases, low pH, or gastric juice (as described in Example 5). It is expected that changes in the hydrodynamic radius will be observed, and the binding of the analyte will be confirmed. Multiple changes in the hydrodynamic range indicate the binding of multiple analytes to the single domain antibody construct, which confirms the formation of higher order complexes. Similarly, this analysis can show how bivalent constructs can achieve increased binding by increasing avidity.

[0421] Example 13: Flow Induced Dispersion Analysis (FIDA)

[0422] Materials and Methods

[0423] FIDA enables the characterization and quantification of proteins under native conditions. FIDA is based on measuring changes in the size of a ligand as it selectively interacts with a target protein. Unbound ligands have a relatively small apparent hydrodynamic radius (size), whereas in the presence of analyte, their apparent hydrodynamic radius increases due to binding to the analyte. Prior to FIDA measurements, V was labeled using the Atto 488 Protein Labeling Kit (Sigma-Aldrich) using a standard protocol. H H antigen (LT-B or FaeG) is fluorescently labeled.

[0424] FIDA quantification of binding between FaeG and F45 monomer and between FaeG and F45::(GGGGS)3::F45 homodivalent protein construct

[0425] FIDA quantification was performed using 39 nL of Atto-labeled FaeG (100 nM) and 7 μL of fermentation medium containing monomers and homodivalent protein constructs. The hydrodynamic radius was detected at 488 nm by detecting the fluorescence of FaeG-atto488. For mixed samples, both monomers and bivalent constructs were pre-incubated with different concentrations of fermentation medium (0 and 50% v / v).

[0426] FIDA quantification of binding between LT-B and LT1 monomer and between LT-B and LT1::(GGGGS)3::LT1 homodivalent protein constructs

[0427] FIDA quantification was performed using 39 nL of Atto labeled LT-B (100 nM) and 7 μL of fermentation medium containing monomers and homodivalent protein constructs. The hydrodynamic radius was detected at 488 nm by detecting the fluorescence of LT-B-atto488. For mixed samples, both monomers and bivalent constructs were pre-incubated with different concentrations of fermentation medium (0-12.5% ​​v / v). LT-specific LT1 monomers and homodivalent protein constructs had been treated with endoH (NEB, P0702) before the experiment to remove known glycosylation.

[0428] FIDA results were analyzed using FIDA-tech software for calculation of binding affinity and visualization of binding curves.

[0429] result

[0430] FaeG

[0431] The hydrodynamic radius of free FaeG was determined, and monomeric F45 (SEQ ID NO: 10) or F45::(GGGGS)3::F45 homodivalent protein construct (SEQ ID NO: 13) was added to assess binding affinity. The hydrodynamic range was determined by measuring the width of the peak produced when the sample was measured using FIDA. Narrow peaks are antigens with no product binding, and peaks become wider (and the hydrodynamic range increases) when product binds. Therefore, higher numbers indicate product binding, and the higher the value, the higher the affinity of the product (see Table 1 below).

[0432] Table 1

[0433] Fermentation medium contents (v / v) Hydrodynamic radius (nm) 0％ 2.40+ / -0.09 50％F45 3.02+ / -0.06 <![CDATA[50%F45::(GGGGS)3::F45]]> 2.97+ / -0.20

[0434] Based on FIDA measurements, we can conclude that both the F45 single domain antibody and the F45::(GGGGS)3::F45 homobivalent protein construct bind FaeG (see Table 1). Interestingly, when correlated with the concentration differences, it can be concluded that the homobivalent protein construct binds with higher affinity than the single domain antibody.

[0435] LT-B

[0436] The hydrodynamic radius of free LT-B was also determined and the binding affinity was assessed by adding LT1 monomer (SEQ ID NO:9) or LT1::(GGGGS)3::LT1 homobivalent protein construct (SEQ ID NO:15) (see Figure 6 ). The bivalent protein construct showed a higher hydrodynamic radius and thus the bivalent protein construct showed a higher binding affinity compared to the single domain antibody.

[0437] in conclusion

[0438] In both experiments, the bivalent protein constructs F45::(GGGGS)3::F45 and LT1::(GGGGS)3::LT1 showed higher binding affinities than the corresponding F45 and LT1 monomers.

[0439] Example 14: F4 + In vivo treatment of post-weaning diarrhea in piglets

[0440] In vivo studies can be performed to determine the effect of the single domain antibody constructs on the treatment of post-weaning diarrhea (PWD) in piglets. + Piglets can be challenged with F4+ETEC bacteria to induce PWD. In the study, 3 technical replicates of 8 piglets can be performed, and each technical replicate is divided into 4 groups of 2 piglets each; one group is control (no treatment), one group will be treated with the single domain antibody construct product, and one group will be treated with F4+ETEC bacteria. + ETEC bacteria stimulation, and the last group will be treated with the single domain antibody construct product and stimulated with F4 + ETEC bacteria. The single domain antibody monomer, construct, the above mixture or any of the above can be tested after treatment with protease, low pH or gastric juice (as described in Example 3). Treatment can be from two days before weaning to 12 days after weaning, and stimulation will be performed on the first and second days after weaning. Disease progression can be determined by measuring analysis of inflammatory biomarkers including degree of diarrhea, body weight, food intake, fever, clinical manifestations and blood samples. Treatment with a single domain antibody construct is expected to reduce or inhibit the development of PWD in all or part of the measured parameters, and may show a curative effect in piglets. This study can be performed multiple times to compare the effects of different combinations of single domain antibody constructs. Therefore, we can compare the progression of PWD in piglets treated with F4 by parameters such as degree of diarrhea, weight gain, food intake and other disease markers relative to untreated piglets. + The in vivo efficacy of the product was demonstrated by improving the performance of piglets challenged with ETEC bacteria.

[0441] Example 15: Use of the same bivalent protein construct to treat F4 + In vivo treatment of post-weaning diarrhoea in piglets Materials and Methods

[0442] Sows (N=5) genetically characterized as susceptible to F4+ Escherichia coli from Aarhus University, Foulum were used for this study and fed a standard Danish sow diet (a diet based on wheat, barley and beans). The piglets were born at AU-Foulum on January 26, 2019 (1st litter = 11 piglets; 2nd litter = 8 piglets, 3rd litter = 5 piglets), 27 days (4th litter = 10 piglets) and 31 days. At weaning (28 days of age), piglets (total N=32) were selected for a stimulation study of F4+ Escherichia coli inoculation. From 3 weeks of age (lactation) to the end of the experiment (5 weeks of age), all pigs were provided with the same feed. Pigs were fed twice a day (semi-free) feed (standard commercial mixture) and provided with free drinking water.

[0443] The experimental design involved four treatments:

[0444] 1) Control: no F45::(GGGGS)3::F45 bivalent protein construct and no F4,

[0445] 2) Control: F45::(GGGGS)3::F45 bivalent protein construct but without F4,

[0446] 3) the same bivalent protein construct without F45::(GGGGS)3::F45 but with F4, and

[0447] 4) There is a bivalent protein construct of F45::(GGGGS)3::F45 and there is F4.

[0448] At weaning, piglets were housed in two penned rooms (with similar climatic conditions) with 2 pigs in each pen for a total of 16 pens, or 4 replicates per group.

[0449] 16 pens were randomly assigned to 2 treatments: solutions of the F45::(GGGGS)3::F45 homobivalent protein construct provided by Bactolife and a control (egg white protein / Novo). These solutions were provided to each pig twice daily starting in the afternoon of the day of weaning and ending 14 days later. The solutions were mixed with non-alcoholic beer (from Hancock) The trial lasted for 21 weeks (until 49 days of age).

[0450] On the 1st and 2nd day after weaning, piglets were challenged orally with Escherichia coli (F4,0149). The CFU of the inoculum at the time of inoculation was 1.05 x 10 on the 1st and 2nd day, respectively. 9and 1.72x10 9 CFU. The control pigs were inoculated with buffer (sodium bicarbonate). Fecal samples (directly from the rectum) were collected from each pig on days 0, 1, 2, 3, 4, 5 and 7 (and every two days in week 2 and every three days in week 3), and the samples were analyzed for hemolytic and non-hemolytic bacteria and verified for Escherichia coli by serological typing. At the end of the trial, the piglets were killed (without sampling).

[0451] result

[0452] An in vivo challenge study was performed on 30 piglets (6-8 per group) in a post-weaning diarrhea model system. The results showed that F4 ETEC was present in the feces of the piglets throughout the experiment. The presence of F4 ETEC in feces indicates an active infection (especially in large numbers). The data generated showed that after the piglets were challenged (days 1+2), F4 ETEC bacteria were present, but they did not establish an infection but were flushed out in piglets treated with the F45::(GGGGS)3::F45 homobivalent protein construct compared to untreated piglets. See Fig.11 .

[0453] in conclusion

[0454] Therefore, it can be concluded that the homobivalent protein construct prevented the adherence of F4 ETEC bacteria in the gastrointestinal tract of newly weaned piglets and significantly reduced the infection time compared to no treatment.

[0455] References

[0456] Gonzales, L.; Ali, ZB; Nygren, E.; Wang, Z.; Karlsson, S.; Zhu, B.;

[0457] Quiding- M.; Alkaline pH Is a Signal for OptimalProduction and Secretion of the Heat Labile Toxin,LT in EnterotoxigenicEscherichia Coli(ETEC).PLoS One 2013,8,e74069

[0458] Harmsen,M.M.;Solt,C.B.Van;Bemmel,A.M.van Z.;Niewold,T.A.;Zijderveld,F.G.van Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy.Appl.Microbiol.Biotechnol.2006,72,544–551,doi:10.1007 / s00253-005-0300-7.

[0459] Harmsen,M.M.;Solt,C.B.Van;Fijten,H.P.D.Enhancement of toxin-andvirus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.Appl.Microbiol.Biotechnol.2009,84,1087–1094,doi:10.1007 / s00253-009-2029-1.

[0460] Lo,A.W.H.;Moonens,K.;De Kerpel,M.;Brys,L.;Pardon,E.;Remaut,H.;DeGreve,H.The molecular mechanism of Shiga toxin Stx2e neutralization byasingle-domain antibody targeting the cell receptor-bindingdomain.J.Biol.Chem.2014,289,25374–25381,doi:10.1074 / jbc.M114.566257.

[0461] Moonens,K.;Kerpel,M.De;Coddens,A.;Cox,E.;Pardon,E.;Remaut,H.;Greve,H.De Nanobody Mediated Inhibition of Attachment of F18 Fimbriae ExpressingEscherichia coli.PLoS One 2014,9,e114691,doi:10.1371 / journal.pone.0114691.

[0462] Van den Broeck,W.;Cox,E.;Godderiis,EMReceptor-DependentImmuneResponses in Pigs after Oral Immunization with F4Fimbriae.Infect.Immun.1999,67,520-526

[0463] Virdi, V.; Coddens, A.; Buck, S.; De; Millet, S.; Maria, B.; Cox, E.; de Greve, H.; de Picker, A. Orally fed seeds producing designer IgAs protect weanedpigletsagainst enterotoxigenic Escherichia coli infection. PNAS 2013,110,11809–11814,doi:10.1073 / pnas.1301975110. Sequence Listing <110> Bactolife ApS <120> Pathogen Binding Protein <130> P5405PC01 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> unknown <220> <223> F41 <220> <221> Peptides <222> (1)..(121) <223> F41 <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Asn Val Asp Arg Ile Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Gly Tyr Ile Ser Glu Gly Gly Ile Leu Asn Tyr Gly Asp Phe Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Asn Leu Lys Ser Glu Asp Thr Gly Val Tyr Phe Cys Ala 85 90 95 Ala Ser His Trp Gly Thr Leu Leu Ile Lys Gly Ile Glu His Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 121 <212> PRT <213> Unknown <220> <223> F42 <220> <221> Peptide <222> (1)..(121) <223> F42 <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Ser Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Ser Lys Arg Glu Phe Val 35 40 45 Ala His Ile Thr Asn Thr Gly Val Thr Glu Phe Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Asp Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Asp Trp Gly Thr Leu Leu Ile Lys Gly Ile Asp His Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 3 <211> 121 <212> PRT <213> Unknown <220> <223> F43 <220> <221> Peptide <222> (1)..(121) <223> F43 <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Asp Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Tyr Val 35 40 45 Ala Ala Ile Ser Trp Thr Gly Ile Ser Thr Tyr Tyr Ala Asp Ile Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Lys Ser Leu Asn Val Pro Ala Pro Trp Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 121 <212> PRT <213> unknown <220> <223> F44 <220> <221> peptide <222> (1)..(121) <223> F44 <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Asp Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Tyr Val 35 40 45 Ala Ala Ile Ser Trp Thr Gly Ile Ser Thr Tyr Tyr Ala Asp Ile Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Arg Ser Leu Asn Val Pro Ala Pro Trp Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 5 <211> 122 <212> PRT <213> unknown <220> <223> Stx2e1 <220> <221> peptide <222> (1)..(122) <223> Stx2e1 <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Arg Leu Ser 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Ser Tyr Gly Asp Thr Asn Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn Ile Glu Ala Gly Thr Tyr Tyr Gly Pro Gly Arg Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 125 <212> PRT <213> Unknown <220> <223> F181 <220> <221> Peptide <222> (1)..(125) <223> F181 <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Gly Arg Asp 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Asp Thr Ser Gly Ile Val Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Ser Ala Lys Lys Thr Leu Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Ser Cys Ala 85 90 95 Thr Gly Pro Phe Val Tyr Gly Arg Gly Cys Leu Gly Gln Ala Phe Tyr 100 105 110 Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 125 <212> PRT <213> Unknown <220> <223> F182 <220> <221> Peptide <222> (1)..(125) <223> F182 <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Arg Lys Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Asn Ser Gly Gly Gly Thr Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asp Thr Val Phe 65 70 75 80 Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Leu Ser Ser Asn Ser Val Cys Pro Pro Gly His Val Ala Trp Tyr 100 105 110 Asn Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 129 <212> PRT <213> Unknown <220> <223> F183 <220> <221> Peptide <222> (1)..(129) <223> F183 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn 20 25 30 Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ser Ile Asn Thr Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Glu Ala Thr Tyr Ser Asp Asn Arg Cys Ser Val Arg 100 105 110 Ser Tyr Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 9 <211> 125 <212> PRT <213> Unknown <220> <223> LT1 <220> <221> Peptide <222> (1)..(125) <223> LT1 <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile 35 40 45 Ser Ser Ile Val Gly Ser Asp Asn Tyr Thr Trp Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Thr Arg Ser Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe 100 105 110 Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 125 <212> PRT <213> Unknown <220> <223> F45 <220> <221> Peptide <222> (1)..(125) <223> F45 <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Val Ser Gly Gly Thr Phe Ser Trp Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Val Ser Arg Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Arg Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 11 <211> 273 <212> PRT <213> Unknown <220> <223> F183::(GGGGS)3::F183 <220> <221> Peptide <222> (1)..(273) <223> F183::(GGGGS)3::F183 <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn 20 25 30 Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ser Ile Asn Thr Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Glu Ala Thr Tyr Ser Asp Asn Arg Cys Ser Val Arg 100 105 110 Ser Tyr Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn 165 170 175 Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 180 185 190 Ala Ser Ile Asn Thr Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 210 215 220 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 225 230 235 240 Ala Ala Val Arg Glu Ala Thr Tyr Ser Asp Asn Arg Cys Ser Val Arg 245 250 255 Ser Tyr Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 260 265 270 Ser <210> 12 <211> 281 <212> PRT <213> Unknown <220> <223> F183::IgG3::F183 <220> <221> Peptide <222> (1)..(281) <223> F183::IgG3::F183 <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn 20 25 30 Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ser Ile Asn Thr Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Glu Ala Thr Tyr Ser Asp Asn Arg Cys Ser Val Arg 100 105 110 Ser Tyr Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Val Asp Ile Glu Pro Pro Thr Pro Ile Cys Pro Glu Ile Cys Ser 130 135 140 Cys Pro Ala Ala Glu Val Leu Gly Gln Val Gln Leu Gln Glu Ser Gly 145 150 155 160 Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170 175 Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Ala Trp Phe Arg Gln Val 180 185 190 Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Asn Thr Arg Gly Gly 195 200 205 Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 210 215 220 Asp Asn Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys Pro 225 230 235 240 Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Ala Val Arg Glu Ala Thr Tyr 245 250 255 Ser Asp Asn Arg Cys Ser Val Arg Ser Tyr Thr Tyr Asp Tyr Trp Gly 260 265 270 Gln Gly Thr Gln Val Thr Val Ser Ser 275 280 <210> 13 <211> 265 <212> PRT <213> Unknown <220> <223> F45::(GGGGS)3::F45 <220> <221> Peptide <222> (1)..(265) <223> F45::(GGGGS)3::F45 <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Val Ser Gly Gly Thr Phe Ser Trp Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Val Ser Arg Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Arg Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 130 135 140 Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 145 150 155 160 Ser Cys Leu Val Ser Gly Gly Thr Phe Ser Trp Tyr Ala Met Gly Trp 165 170 175 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Thr Val Ser 180 185 190 Arg Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 195 200 205 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 210 215 220 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Arg 225 230 235 240 Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 14 <211> 259 <212> PRT <213> Unknown <220> <223> Stx2e1::(GGGGS)3::Stx2e1 <220> <221> Peptide <222> (1)..(259) <223> Stx2e1::(GGGGS)3::Stx2e1 <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Arg Leu Ser 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Ser Tyr Gly Asp Thr Asn Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn Ile Glu Ala Gly Thr Tyr Tyr Gly Pro Gly Arg Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser 130 135 140 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 145 150 155 160 Val Ser Gly Ser Ile Phe Arg Leu Ser Thr Met Gly Trp Tyr Arg Gln 165 170 175 Ala Pro Gly Lys Gln Arg Glu Phe Val Ala Ser Ile Thr Ser Tyr Gly 180 185 190 Asp Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 195 200 205 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 210 215 220 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Asn Ile Glu Ala Gly Thr 225 230 235 240 Tyr Tyr Gly Pro Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser <210> 15 <211> 265 <212> PRT <213> Unknown <220> <223> LT1::(GGGGS)3::LT1 <220> <221> Peptide <222> (1)..(265) <223> LT1::(GGGGS)3::LT1 <400> 15 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile 35 40 45 Ser Ser Ile Val Gly Ser Asp Asn Tyr Thr Trp Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Thr Arg Ser Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe 100 105 110 Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 130 135 140 Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 145 150 155 160 Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Ile Gly Trp 165 170 175 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile Ser Ser Ile Val 180 185 190 Gly Ser Asp Asn Tyr Thr Trp Tyr Thr Asp Ser Val Lys Gly Arg Phe 195 200 205 Thr Ile Ser Ser Asp Asn Thr Arg Ser Thr Val Tyr Leu Gln Met Asn 210 215 220 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Ser 225 230 235 240 Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe Gly Ser Trp Gly 245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 16 <211> 265 <212> PRT <213> Unknown <220> <223> F45::(GGGGS)3::LT1 <220> <221> Peptide <222> (1)..(265) <223> F45::(GGGGS)3::LT1 <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Val Ser Gly Gly Thr Phe Ser Trp Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Val Ser Arg Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Arg Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 130 135 140 Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 145 150 155 160 Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Ile Gly Trp 165 170 175 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile Ser Ser Ile Val 180 185 190 Gly Ser Asp Asn Tyr Thr Trp Tyr Thr Asp Ser Val Lys Gly Arg Phe 195 200 205 Thr Ile Ser Ser Asp Asn Thr Arg Ser Thr Val Tyr Leu Gln Met Asn 210 215 220 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Ser 225 230 235 240 Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe Gly Ser Trp Gly 245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 17 <211> 265 <212> PRT <213> Unknown <220> <223> LT1::(GGGGS)3::F45 <220> <221> Peptide <222> (1)..(265) <223> LT1::(GGGGS)3::F45 <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile 35 40 45 Ser Ser Ile Val Gly Ser Asp Asn Tyr Thr Trp Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Thr Arg Ser Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe 100 105 110 Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 130 135 140 Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 145 150 155 160 Ser Cys Leu Val Ser Gly Gly Thr Phe Ser Trp Tyr Ala Met Gly Trp 165 170 175 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Thr Val Ser 180 185 190 Arg Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 195 200 205 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 210 215 220 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Arg 225 230 235 240 Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 18 <211> 266 <212> PRT <213> Unknown <220> <223> F183::(GGGGS)3::Stx2e1 <220> <221> Peptide <222> (1)..(266) <223> F183::(GGGGS)3::Stx2e1 <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn 20 25 30 Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ser Ile Asn Thr Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Glu Ala Thr Tyr Ser Asp Asn Arg Cys Ser Val Arg 100 105 110 Ser Tyr Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Arg Leu Ser 165 170 175 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 180 185 190 Ala Ser Ile Thr Ser Tyr Gly Asp Thr Asn Tyr Arg Asp Ser Val Lys 195 200 205 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 210 215 220 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 225 230 235 240 Ala Asn Ile Glu Ala Gly Thr Tyr Tyr Gly Pro Gly Arg Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 19 <211> 266 <212> PRT <213> Unknown <220> <223> Stx2e1::(GGGGS)3::F183 <220> <221> Peptide <222> (1)..(266) <223> Stx2e1::(GGGGS)3::F183 <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Arg Leu Ser 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Ser Tyr Gly Asp Thr Asn Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn Ile Glu Ala Gly Thr Tyr Tyr Gly Pro Gly Arg Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser 130 135 140 Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 145 150 155 160 Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Ala Trp Phe Arg Gln 165 170 175 Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Asn Thr Arg Gly 180 185 190 Gly Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 195 200 205 Arg Asp Asn Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys 210 215 220 Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Ala Val Arg Glu Ala Thr 225 230 235 240 Tyr Ser Asp Asn Arg Cys Ser Val Arg Ser Tyr Thr Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> GGGGS Linker <220> <221> Peptide <222> (1)..(5) <223> GGGGS Linker <400> 20 Gly Gly Gly Gly Ser 1 5 <210> twenty one <211> 10 <212> PRT <213> Artificial sequence <220> <223> (GGGGS)2 connector <220> <221> Peptides <222> (1)..(10) <223> (GGGGS)2 connector <400> twenty one Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> twenty two <211> 15 <212> PRT <213> Artificial sequence <220> <223> (GGGGS)3 connector <220> <221> Peptides <222> (1)..(15) <223> (GGGGS)3 connector <400> twenty two Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> twenty three <211> 20 <212> PRT <213> Artificial sequence <220> <223> (GGGGS)4 connector <220> <221> Peptides <222> (1)..(20) <223> (GGGGS)4 connector <400> twenty three Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 24 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> (GGGGS)5 linker <220> <221> Peptide <222> (1)..(25) <223> (GGGGS)5 linker <400> 24 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 25 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> (GGGGS)6 linker <220> <221> Peptide <222> (1)..(30) <223> (GGGGS)6 linker <400> 25 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 26 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> IgG3 linker <220> <221> Peptides <222> (1)..(23) <223> IgG3 linker <400> 26 Val Asp Ile Glu Pro Pro Thr Pro Ile Cys Pro Glu Ile Cys Ser Cys 1 5 10 15 Pro Ala Ala Glu Val Leu Gly 20 <210> 27 <211> 7 <212> PRT <213> unknown <220> <223> LT1 CDR1 <220> <221> Peptides <222> (1)..(7) <223> LT1 CDR1 <400> 27 Phe Thr Phe Asp Asp Tyr Ala 1 5 <210> 28 <211> 9 <212> PRT <213> unknown <220> <223> F45 CDR1 <220> <221> Peptides <222> (1)..(9) <223> F45 CDR1 <400> 28 Gly Thr Phe Ser Trp Tyr Ala Met Gly 1 5 <210> 29 <211> 9 <212> PRT <213> unknown <220> <223> LT1 CDR2 <220> <221> Peptides <222> (1)..(9) <223> LT1 CDR2 <400> 29 Ser Ile Val Gly Ser Asp Asn Tyr Thr 1 5 <210> 30 <211> 11 <212> PRT <213> unknown <220> <223> F45 CDR2 <220> <221> Peptides <222> (1)..(11) <223> F45 CDR2 <400> 30 Thr Val Ser Arg Gly Gly Gly Ser Ser Tyr Tyr 1 5 10 <210> 31 <211> 18 <212> PRT <213> unknown <220> <223> LT1 CDR3 <220> <221> Peptides <222> (1)..(18) <223> LT1 CDR3 <400> 31 Ala Ala Asp Ser Tyr Ser Asp Tyr Val Ser Leu Asn Pro Glu Thr Phe 1 5 10 15 Gly Ser <210> 32 <211> 18 <212> PRT <213> unknown <220> <223> F45 CDR3 <220> <221> Peptides <222> (1)..(18) <223> F45 CDR3 <400> 32 Gly Arg Gly Ala Pro Ser Asp Thr Gly Arg Pro Asp Glu Tyr Asp Tyr 1 5 10 15 Trp Gly

Claims

1. An isolated protein consisting of - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein said linker is not an IgG3 linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, in, The first peptide and the second peptide are single domain antibodies against the B subunit of the heat-labile toxin, the first peptide comprises three complementarity determining regions: CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:31, and wherein the second peptide comprises three complementarity determining regions: CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:31; or in, The first peptide and the second peptide are single-domain antibodies against FaeG, the first peptide comprising three complementarity determining regions: CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:32, and wherein the second peptide comprises three complementarity determining regions: CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:

32.

2. The protein of claim 1, wherein the protein is stable in the gastrointestinal tract of a subject.

3. The protein of claim 1, wherein the protein is stable in the respiratory system of a subject.

4. The protein of claim 1, wherein the protein is acid stable.

5. The protein of claim 1, wherein the protein is protease stable.

6. The protein of claim 1, wherein the protein is pH stable.

7. The protein of claim 1, wherein the protein is temperature stable.

8. The protein of claim 1, wherein the linker is a GS linker.

9. The protein of claim 8, wherein the GS linker has (G x S) n Structure, where x is a number between 1 and 10, and n refers to G x The number of repetitions of the S sequence, where n is between 1 and 10.

10. The protein of claim 9, wherein x is 2 to 5.

11. The protein of claim 9, wherein n is 2 to 5.

12. The protein of claim 8, wherein the GS linker is a GGGGS linker (SEQ ID NO: 20), a GGGGSGGGGS linker (SEQ ID NO: 21), a GGGGSGGGGSGGGGS linker (SEQ ID NO: 22), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 23), a GGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 24), or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 25).

13. The protein of claim 1, with the proviso that the linker does not comprise a portion of an antibody.

14. The protein of claim 13, wherein the portion of the antibody is an Fc region or a portion of an Fc region.

15. The protein of claim 1, wherein the binding affinity of the protein is at least 1.5 times higher than the binding affinity of a monomeric single domain antibody.

16. The protein of claim 1, wherein the peptide binds with low affinity to non-pathogen surface components and / or molecules secreted by non-pathogens.

17. The protein of claim 1, wherein the first peptide and the second peptide both comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

10.

18. The protein of any one of the preceding claims 1-17, wherein the first peptide and the second peptide both comprise the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

19. The protein of any one of claims 1 to 17, wherein the protein comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO:

15.

20. An isolated nucleic acid molecule encoding the protein of any one of claims 1 to 19.

21. A vector comprising the nucleic acid molecule of claim 20.

22. The vector of claim 21, wherein the vector is an expression vector.

23. A recombinant host cell comprising the nucleic acid molecule of claim 20 or the vector of claim 21 or 22.

24. The recombinant host cell of claim 23, wherein the host cell is a bacterial, fungal or mammalian cell.

25. The recombinant host cell of claim 24, wherein the fungus is a yeast.

26. The recombinant host cell of claim 24, wherein the bacterium is Bacillus.

27. The recombinant host cell of claim 26, wherein the bacterium is Bacillus licheniformis, Bacillus subtilis, or Bacillus lactobacillus.

28. The recombinant host cell of claim 25, wherein the yeast is selected from the genera Pichia, Hansenula, and Saccharomyces.

29. The recombinant host cell of claim 24, wherein the fungus is selected from Aspergillus oryzae and Aspergillus niger.

30. A composition comprising the protein of any one of claims 1 to 19.

31. Use of the composition of claim 30 for promoting growth in a subject, wherein the use is non-therapeutic.

32. A pharmaceutical composition comprising a protein as described in any one of claims 1 to 19, a nucleic acid molecule as described in claim 20, a vector as described in any one of claims 21 to 22, and / or a recombinant host cell as described in any one of claims 23 to 29, and further comprising one or more excipients.

33. The pharmaceutical composition of claim 32, wherein the excipient is selected from the group consisting of surfactants, antiadherents, binders, coating agents, disintegrants, fillers, solvents / cosolvents, flavoring agents, pigments, lubricants, glidants, preservatives, adsorbents, sweeteners, carriers, polymers, buffers, antioxidants, wetting agents, defoamers, thickeners, wetting agents, and mixtures thereof.

34. The pharmaceutical composition of claim 32, further comprising one or more carbohydrates and / or alginates, enzymes, activated carbon, antibiotics, and mixtures thereof.

35. The pharmaceutical composition of claim 34, wherein the carbohydrate is cellulose.

36. Use of a protein as described in any one of claims 1 to 19, a nucleic acid molecule as described in claim 20, a vector as described in any one of claims 21 to 22, a recombinant host cell as described in any one of claims 23 to 29 or a pharmaceutical composition as described in any one of claims 32 to 35 in the preparation of a medicament for preventing or treating gastrointestinal infections.

37. The use of claim 36, wherein the route of administration of the drug is oral.

38. The use of claim 36, wherein the use reduces the time span of active infection.

39. The use of claim 36, wherein said use reduces toxicity.

40. The use of claim 36, wherein the subject is a human subject.

41. The use of claim 36, wherein the subject is a livestock animal.

42. The use of claim 41, wherein the livestock animal is an animal selected from the group consisting of pigs, cattle, sheep, goats, horses, chickens, donkeys, mules, ducks, geese and turkeys.

43. The use of claim 36, wherein the subject is a pig.

44. Use of a protein as claimed in any one of claims 1 to 19, a nucleic acid molecule as claimed in claim 20, a vector as claimed in any one of claims 21 to 22, a recombinant host cell as claimed in any one of claims 23 to 29, or a pharmaceutical composition as claimed in any one of claims 32 to 35, in the preparation of a medicament for preventing or treating post-weaning diarrhea (PWD).

45. Use of a protein as described in any one of claims 1 to 19, a nucleic acid molecule as described in claim 20, a vector as described in any one of claims 21 to 22, a recombinant host cell as described in any one of claims 23 to 29, or a pharmaceutical composition as described in any one of claims 32 to 35 in the preparation of a medicament for preventing or treating porcine edema disease.

46. ​​A method for producing the protein of any one of claims 1 to 19, the method comprising a) transfecting the nucleic acid molecule according to claim 20 and / or the vector according to any one of claims 21 to 22 into a host cell to obtain a recombinant host cell according to any one of claims 23 to 29; b) culturing the recombinant host cell under appropriate conditions; c) collecting and purifying the protein expressed from the recombinant host cell.

Images