Single-domain antibodies to reduce the risk of cholera and e. coli infection

Abstract

A single-domain antibody directed against Vibrio cholerae and toxin, particularly subunit B of Vibrio cholerae toxin.

Description

[0001] Single-domain antibodies to reduce the risk of Cholera and E. coll infection

[0002] Field of the invention

[0003] The present disclosure relates to a single-domain antibody directed against Vibrio cholerae toxin, particularly subunit B of Vibrio cholerae toxin.

[0004] Background of the invention

[0005] As known in the art - a nanobody or single domain antibody (sdAb) refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.

[0006] Such single domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian host cell).

[0007] Cholera is caused by the Gram-negative bacterium, Vibrio cholerae, often spreading via contaminated water or foods. The bacterium can colonize the small intestine and produce enterotoxins, such as the main virulence factor cholera toxin (CT), causing severe diarrhoea. There are more than a billion people living at risk of cholera worldwide and 140,000 associated deaths each year, a third of all deaths are children under the age of five.

[0008] CT consists of two subunits, the A-subunit (CTA) and B-subunit (CTB). CTB enables toxin transfer across the outer membrane of intestinal cells in the gut through interaction with specific ganglioside receptors (GM1). Once intracellular, CTA initiates a G-protein coupled cascade that ultimately causes fluid secretion into the intestinal lumen.

[0009] As described in Harmsen et al. (Appl Microbiol Biotechnol (2009) 84:1087-1094) - heat-labile enterotoxin (LT) secreted by enterotoxigenic Escherichia coll (ETEC) may be seen as related to CT.

[0010] Both LT and CT are related to diarrheal diseases.

[0011] Enterotoxigenic Escherichia coll (ETEC) causes millions of diarrheal episodes each year and is the most common bacterial cause of diarrhea for children in low- and middle-income countries (LMICs).

[0012] Harmsen discloses a single domain antibody (sdAb) termed “LT109” and provides result showing that this LT109 in / V-glycosylated form (recombinantly expressed from S. cerevisiae) had best binding and blocking of tested sdAbs to LT - the article reads:

[0013] [page 1089, right column, last paragraph]: “Most neutralizing clones also inhibited binding of LT to its cellular receptor GM1 , as determined by ELISA. Clone LT109 again had the lowest IC50 (Table 1).” [page 1091 , left column]:

[0014] “... / V-glycosylation of LT109 contributes to its LT-neutralizing capacity”.

[0015] The article of Harmsen also describes a qualitative test of binding to CTB - page 1089, right column, last paragraph reads:

[0016] “Most clones cross-reacted sufficiently to CT holotoxin to enable such determination (Table 1)... suggesting that they bind the B-subunit.”.

[0017] Accordingly, with respect to CT binding - the article only makes a qualitative statement relating to possible suggested CTB binding - i.e. the article provides no statement relating to a quantitatively possible better binding of one sdAb (e.g. LT109) over another clone (sdAb).

[0018] The LT109 sdAb disclosed in Harmsen is a monomer.

[0019] WO2020 / 144164A1 (Bactolife) describes a homodimer of LT109 - termed “LT1 ::(GGGGS)3::LT1 ” (see e.g. Table 1 of page 42) and provides results showing that the homodimer binds to LT with a higher binding affinity as compared to the corresponding monomer (see e.g. Conclusion of Example 13 on page 56) - i.e. homodimer “LT1 ::(GGGGS)3::LT1 ” (may herein be termed “BP-L”) binds to LT with a higher binding affinity as compared to the LT 109 monomer clone / sdAb of Harmsen.

[0020] WO2020 / 144164A1 (Bactolife) reads on page 30: “the invention concerns a use of the protein or composition for prevention or treatment of infection caused by Vibrio cholera ".

[0021] No experimental data is provided with respect to any possible CT binding of any of the several different sdAbs discussed in WO2020 / 144164A1 - i.e. it is a mere general statement.

[0022] Taheri et al. (International Immunopharmacology 89 (2020) 107054) discloses protective effects of egg yolk IgYs developed against recombinant immunogens CtxB, OmpW and Tcpa on infant mice infected with Vibrio cholerae.

[0023] Barati et al. (Iran J Immunol. 2018; 15(1):47-58) teaches the production of chicken egg yolk antibody IgY against recombinant cholera toxin B subunit.

[0024] Garcia et al. (Scientific Reports, vol. 13, no. 1 , 15 March 2023) relates to monoclonal antibodies to cholera toxin B subunit, in particular the antibody 7A12B3 that was found to be a potent inhibitor of cholera holotoxin (CTX).

[0025] Legler et al. (Acta Cryst. (2013). F69, 90-93) teaches the structure of a low-melting-temperature anticholera toxin with llama VHH domain. Summary of the invention

[0026] A problem to be solved by the present invention relates to the identification of a single domain antibody (sdAb) related construct / product that may be used to neutralize the pathogenicity of both enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae.

[0027] As discussed above - CTB enables toxin transfer across the outer membrane of intestinal cells in the gut through interaction with specific ganglioside receptors (GM1).

[0028] Accordingly, to properly be able to neutralize the pathogenicity of Vibrio cholerae - it is not enough that the antibody has some binding to the CT, since it need to be able in a clinical useful way to block CT-GM1 receptor interaction.

[0029] As discussed in further detail in Example 1 herein - the present inventors tested several antibodies having strong binding to CTB and they only displayed a modest blocking capacity of CTB-GM1 interaction.

[0030] As discussed above - the article of Harmsen only make a qualitative statement relating to possible suggested CTB binding - i.e. the article provides no herein relevant experimental information with respect to a possible significantly blocking of the CT-GM1 receptor interaction of any of the antibodies tested in Harmsen.

[0031] Despite the results of Example 1 , which may be seen as demonstrating it is difficult to identify an antibody that significantly the block CT-GM1 receptor interaction - the present inventors continued their work and identified that the above discussed homodimer “LT1 ::(GGGGS)3::LT1 ” (may herein be termed “BP-L”) disclosed in WO2020 / 144164A1 has a surprisingly good capacity of blocking of the CT- GM1 receptor interaction (see Example 2).

[0032] The “(GGGGS)3” in “LT1 ::(GGGGS)3::LT1 ” (BP-L) is a GS linker sequence - and as understood by the skilled person - there may be used other suitable linker sequences.

[0033] Without being limited to theory - it is believed that the results of e.g. the working examples herein (e.g. Example 2) make it plausible that “LT1 ” monomer sdAb would as such also have a relevant capacity of blocking of the CT-GM1 receptor interaction.

[0034] Further, once having demonstrated as done herein that “LT1” has a relevant capacity of blocking of the CT-GM1 receptor interaction - the skilled person may use this new information to make relatively minor changes in e.g. the CDR regions, while maintaining a relevant capacity of blocking of the CT- GM1 receptor interaction. See e.g. Example 5 herein for a further discussion of this matter.

[0035] In WO2020 / 144164A1 (Bactolife): - LT1 : CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 29 and CDR3 is SEQ ID NO: 31 ;

[0036] - LT1 is SEQ ID NO: 9;

[0037] - “LT1 ::(GGGGS)3::LT1” (BP-L) is SEQ ID NO: 15.

[0038] In the present application the corresponding SEQ ID NO numbers are:

[0039] - LT1 : CDR1 is SEQ ID NO: 1 , CDR2 is SEQ ID NO: 2 and CDR3 is SEQ ID NO: 3;

[0040] - LT1 is SEQ ID NO: 4;

[0041] - “LT1 ::(GGGGS)3::LT1” (BP-L) is SEQ ID NO: 5.

[0042] Accordingly, a first aspect of the present invention relates to a single-domain antibody (sdAb) which binds to subunit B of Vibrio cholerae toxin (CT), wherein said single-domain antibody is comprising:

[0043] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and

[0044] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and

[0045] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; for use in the reduction of risk, prevention or treatment of Vibrio cholerae infection in a subject.

[0046] As discussed above - cholera is caused by the Gram-negative bacterium Vibrio cholerae.

[0047] Accordingly, the term “Vibrio cholerae infection” of the first aspect may alternatively be termed “cholera”, which is known to be a diarrheal disease.

[0048] The term “treatment” encompasses both curative and ameliorative treatment. By ameliorative treatment is meant a treatment that results in the improvement of one or more symptoms of a herein relevant infection in a subject.

[0049] Embodiments of the present invention are described below, by way of examples only.

[0050] Drawing description

[0051] Figure 1 : Shows BP-L (0.1-250 nM) neutralization of CTX toxicity (0.230 nM) against a mammalian intestinal cell line (HCA-7) at increasing concentrations. See Example 4 herein for further details.

[0052] Figure 2. Shows a sequence analysis of BP-L in relation to CDR regions (CDR1-3) highlighted in grey and crucial amino acid residues in these regions (light grey) with regards to CTB-binding, based on in silico (ColabFold) predictions of protein-protein interactions. See Example 5 herein for further details. The sequence of Figure 2 is SEQ ID NO: 4 herein.

[0053] Detailed description of the invention

[0054] For the skilled person it is routine work to obtain a suitable single-domain antibody (sdAb) in relation to a suitable pathogen of interest - i.e. it is herein not considered necessary to discuss this in great details.

[0055] As discussed above, and as known in the art - a nanobody or single domain antibody (sdAb) refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.

[0056] Such single domain antibodies can be derived from antibodies raised in e.g. sharks or e.g. Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian host cell).

[0057] As discussed above - in relation to herein relevant sdAbs - suitable methods for obtaining / producing them are e.g. described in Harmsen and WO2020 / 144164A1 (Bactolife).

[0058] Preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein said single-domain antibody is comprising:

[0059] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein 1 amino acid has been altered; and

[0060] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein 1 amino acid has been altered; and

[0061] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein 1 amino acid has been altered.

[0062] More preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein said single-domain antibody is comprising:

[0063] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and

[0064] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and

[0065] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3.

[0066] This embodiment is termed “embodiment CDR1-3 = SEQ ID NO: 1-3” herein.

[0067] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

[0068] In one embodiment, the single-domain antibody of the present disclosure is a humanized version of the single-domain antibody of the first aspect and / or embodiment thereof.

[0069] The present disclosure also provides fusion proteins comprising at least one of the single-domain antibodies as described herein. Such fusion protein can be assembled by methods known to the person skilled in the art. Preferably, the fusion protein is recombinantly designed by fusing gene sequences in vitro. In some embodiments, the fusion protein further comprises a linker connecting the sbAbs.

[0070] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is a fusion protein comprising a single-domain antibody as defined in the first aspect and / or embodiment thereof and one or more further single-domain antibodies as defined in the first aspect and / or embodiment thereof, and optionally one or more linkers, such as one or more GS linkers.

[0071] In a preferred embodiment the fusion protein comprises a linker connecting the sdAbs.

[0072] In one embodiment the linker is a GS linker, i.e. a linker which comprises or consists of glycine and serine residues. Such linkers are well known in the art.

[0073] In one embodiment the linker is a GS linker of the structure (GxS)n, where x may be a number between 1 to 10, preferably 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 10, preferably 2 to 5.

[0074] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the fusion protein is a homodimer or a heterodimer - more preferably the fusion protein is a homodimer.

[0075] In a preferred embodiment, the fusion protein homodimer is comprising a single-domain antibody as defined in “embodiment CDR1-3 = SEQ ID NO: 1-3” discussed above and one more single-domain antibody as defined in “embodiment CDR1-3 = SEQ ID NO: 1-3” discussed above.

[0076] Preferably, the fusion protein homodimer is comprising or consisting of the sequence as set forth in SEQ ID NO: 5, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs. More preferably, the fusion protein homodimer is comprising or consisting of the sequence as set forth in SEQ ID NO: 5.

[0077] As discussed above, herein relevant “LT1 ” related sdAbs are in the prior art described to also bind to heat-labile enterotoxin (LT) secreted by enterotoxigenic Escherichia coli (ETEC).

[0078] Accordingly, in a preferred embodiment - the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody also binds to heat-labile enterotoxin (LT) secreted by enterotoxigenic Escherichia coli (ETEC) and wherein the single-domain antibody is also for use in the reduction of risk, prevention or treatment of enterotoxigenic Escherichia coli (ETEC) infection in a subject.

[0079] As discussed above, both LT and CT toxins are related to diarrheal diseases - i.e. a sdAb as discussed herein may be used to neutralize the pathogenicity of both enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in a subject (e.g. a human) - which is a significant clinically relevant advantage.

[0080] In relevant working examples herein were used a glycosylated single-domain antibody and it gave surprisingly good results (see e.g. Example 2).

[0081] Accordingly, in a preferred embodiment - the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is a glycosylated single-domain antibody.

[0082] The skilled person knows how to produce / obtain a herein relevant glycosylated single-domain antibody - e.g. by recombinant expression in a suitable eucaryotic production host cell (e.g. yeast, Aspergillus, etc.).

[0083] In one embodiment, the production host cell is a yeast, such as a yeast selected from the genus of Pichia (preferably Pichia pastoris), Hansenula or Saccharomyces.

[0084] As known in the art - Pichia may alternatively be named Komagataella and Pichia pastoris may alternatively be named Komagataella phaffii.

[0085] In one embodiment, the production host cell is a fungus selected from Aspergillus - such as e.g. Aspergillus oryzae or Aspergillus niger.

[0086] Example 1 of WO2020 / 144164A1 (Bactolife) describes recombinant expression of sdAb in Aspergillus oryzae, and this was also used in working examples herein with very good results. Accordingly, in a preferred embodiment the glycosylated single-domain antibody had been obtained by recombinant expression in Aspergillus oryzae - wherein the sdAb is preferably a fusion protein homodimer antibody as discussed above.

[0087] A subject may e.g. be an animal or a human - preferably, the subject is a human.

[0088] In one embodiment, the subject is a human child, such as a child of less than 10 years of age, and / or an immunocompromised subject. In one embodiment the subject is a child of between 0 and 10 years of age, such as a child of between 1 and 6 years of age, such as a child of between 1 and 5 years of age, such as a child of between 1 and 4 years of age, such as a child of between 2 and 5 years of age, such as a child of between 2 and 4 years of age.

[0089] As discussed above - the present inventors identified that herein relevant sdAb has a surprisingly good capacity of blocking of the CT-GM1 receptor interaction (see e.g. Example 2).

[0090] Accordingly, in a preferred embodiment - the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody (sdAb) is capable of blocking the CT-GM1 receptor interaction with a blocking capacity of at least 50% (preferably at least 70%, more preferably at least 90%, and most preferably at least 98%) at sdAb:CTB binding site ratio of 2:1 , measured according to working Example 1 herein.

[0091] As understood by the skilled person - a 1 :1 molar ratio for a homodimer, that comprises two antigen binding sites, corresponds to a 2:1 sdAb:CTB binding site ratio.

[0092] Due to the good capability of blocking the CT-GM1 receptor interaction - one may use a relatively low dose.

[0093] Preferably, the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is present in a dietary composition or a pharmaceutical composition.

[0094] According to the art - for pharmaceutical compositions, all components of the composition should be pharmaceutically acceptable. By “pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the sbAb. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)).

[0095] In one embodiment, the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and / or nutrients, such as dietary minerals. In one embodiment, the dietary composition is an oral rehydration solution or yoghurt.

[0096] EXAMPLES

[0097] The examples below refer to below mentioned materials and methods related references - these references are as such not related to the inventive concept of the present invention.

[0098] - Ahmadi et al. (Scientific Reports volume 10, Article number: 10765 (2020))

[0099] EXAMPLE 1 : Comparative Example - “Binding to CT’ - “Blocking of the CT-GM1 receptor interaction”

[0100] Binding to cholera toxin (CT)

[0101] Monomeric sdAb-containing Escherichia coll supernatants selected from phage display screenings (second and third panning round) were evaluated based on their CTB binding capacity in a direct enzyme-linked immunosorbent assay (ELISA), similar to the experimental approach described by Ahmadi et al. (2020). In essence, streptavidin 10 pg / ml was used to capture 60 pL of biotinylated CTB at 5 pg / mL. Afterwards, E. coll expressing sdAbs were replicated in auto-induction media and their supernatants (30 pL) tested for binding against the biotinylated antigen in MaxiSorp plates. A 20,000- fold dilution of Monoclonal Anti-FLAG M2-peroxidase covalently conjugated to horseradish peroxidase (Sigma, A8592) was used to detect sdAb-CTB binding.

[0102] Blocking of the CT-GM1 receptor interaction:

[0103] The blocking capacity of the monomeric single-domain antibodies in E. coll supernatants selected based on their affinity towards CTB, were purified and further evaluated for their blocking capacity of CTB interaction with the intestinal GM1 receptor. Their blocking capacity were determined using a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). Briefly, 180 nM of each singledomain antibody was incubated with 18 nM biotinylated CTB, before being added to a black 96-well Immuno Plate previously coated with 5 ug / ml of GM1 receptor. Biotinylated CTB (18 nM) incubated at 37 °C was used as a reference for the blocking effect.

[0104] Streptavidin-conjugated europium (Perkin Elmer, #1244-360) diluted 1 / 500 in DELFIA assay buffer (Perkin Elmer #1244-111) was added and the plate was incubated for 30 minutes at room temperature. DELFIA enhancement solution 20 (Perkin Elmer #4001-0010) was used to activate europium fluorescence, followed by intensity measurements at 615 nm.

[0105] The single-domain antibodies selected for their high CTB affinity, demonstrated a modest blocking capacity of CTB interaction with the GM1 receptor at a 10:1 molar ratio (sdAb:CTB).

[0106] Results

[0107] The table below shows results of “binding to CT” and “Blocking of the CT-GM1 receptor interaction” for different sdAbs originally selected from CTB-specific phage display screening.

[0108] Conclusions

[0109] The result of this example shows that the tested antibodies selected from a CTB-specific monomeric sdAb library have a strong binding affinity towards CTB but display a modest blocking capacity of CTB- GM1 interaction.

[0110] EXAMPLE 2: “LT1 ::(GGGGS)3::LT1” (“BP-L”) disclosed in W02020 / 144164A1 - surprisingly good capacity of blocking of the CT-GM1 receptor interaction

[0111] As discussed above - “LT1 ::(GGGGS)3::LT1 ” (BP-L) is SEQ ID NO: 5 herein.

[0112] Recombinant expression of BP-L in Aspergillus oryzae was done as described in WO2020 / 144164A1 (Bactolife) and glycosylated BP-L was obtained.

[0113] Testing for blocking of the CTB-GM1 receptor interaction was done as described in Example 1 .

[0114] Results

[0115] The table below shows results of “Blocking of the CT-GM1 receptor interaction” for glycosylated homodimeric BP-L.

[0116] Conclusions

[0117] The results of this example show that “BP-L” has a surprisingly good capacity of blocking of the CTB- GM1 receptor interaction.

[0118] In fact - the BP-L capacity of blocking of the CTB-GM1 receptor interaction was significantly better than any of the different sdAbs tested in Example 1 .

[0119] EXAMPLE 3: “LT1::(GGGGS)3::LT1” (“BP-L") disclosed in W02020 / 144164A1 - affinity to LT and CTB

[0120] LTB and CTB affinity determination.

[0121] The apparent affinity (KD) of BP-L towards heat-labile enterotoxin (LT) secreted by enterotoxigenic Escherichia coli (ETEC) and CTB was determined using biolayer interferometry on an Octet RED96 instrument (ForteBio). At first, 76 nM of biotinylated BP-L was captured on a streptavidin biosensor (Satorius, 18-5020) before it was exposed to a serial dilution (120 to 0.470 nM) of antigen (e.g., CTB) in running buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 50 mM MES hydrate, 0.05% P20, pH 7.24). Antigen association (Kon) and dissociation (KOff) were measured for 600 s, respectively. The control biosensor signal, obtained from subjecting biotinylated BP-L to running buffer alone, was subtracted and the affinity calculated using the Octet Analysis Studio (ForteBio).

[0122] Results

[0123] The table below shows the binding affinity (KD) of BP-L towards LT and CTB.

[0124] *The Octet RED96 has a detection limit (KD) of 1 .OE-12 M.

[0125] *As known in the art - LTB is the GM1 binding subunit of LT

[0126] Conclusions

[0127] The results of this example show that BP-L has a surprisingly strong binding affinity towards both CTB and LTB, with binding affinity towards the latter stronger than the instruments limit of detection.

[0128] EXAMPLE 4: BP-L effectively neutralizes CTX toxicity in an intestinal cell model Uptake of CTX by endothelial cells of the small intestine induces increased levels of cellular cAMP. The ability of BP-L to neutralize the cytotoxic activity of CTX was determined using a human HCA-7 colon cancer cell line (AddexBio, C0009003). CTX (0.230 nM) was incubated with different concentrations of BP-L (0.1-250 nM) before being added to 1 x 104HCA-7 cells per well. The amount of intracellular cAMP was detected after 2 hours according to the manufacturer’s protocol (cAMP-Glo™ Max Assay, Promega, Madison, Wl) and luminescence measured using a Victor Nivo Multimode plate reader. Blocking capacity was determined in relation to a CTX-only reference. The experiment was analyzed in duplicates of technical triplicates.

[0129] Results

[0130] Figure 1 shows that BP-L was able to block all cellular uptake of CTX at a 1 :135 (CTX:BP-L) molar ratio.

[0131] Conclusions

[0132] The results of this example show that BP-L is a highly effective blocker of CTX interaction with GM1 receptors on the cell surface of human intestinal epithelia.

[0133] EXAMPLE 5: Paratope analysis of BP-L using open access software

[0134] The protein-protein interaction between BP-L and CTB was predicted using ColabFold in combination with classical molecular dynamics simulations. Starting from previously published data on CTB-GM1 interactions, three repetitions of 1000 ns of classical molecular dynamics simulations were performed, using the AMBER 24 simulation software containing the pmemd.cuda module.

[0135] The structure models were placed into cubic water boxes of TIP3P water molecules with a minimum wall distance to the protein of 12 A. Parameters for all simulations were derived from the AMBER force field MSB. Bonds involving hydrogen atoms were restrained using the SHAKE algorithm, allowing a timestep of 2.0 femtoseconds. System pressure was maintained at 1 bar by applying weak coupling to an external bath using the Berendsen algorithm. The Langevin Thermostat was utilized to keep the temperature at 300K during the simulations.

[0136] The obtained MD simulations for BP-L in complex with CTB were used to calculate contacts using the GetContacts software. In addition, CPPTRAJ was utilized for hierarchical agglomerative clustering analysis to identify the most representative binding poses.

[0137] Results

[0138] Figure 2 shows an in silico paratope analysis (ColabFold) of BP-L interaction with CTB, in comparison to previously published sequence information on the CTB epitope participating in the GM1 receptor interaction. This data indicates that out of all amino acids in the three CDR regions (CDR1-3), there are 10 amino acid residues (D55, D99, S100, Y101 , D103, Y104, V105, S106, E110, and T111) in the BP-L paratope predicted to be crucial for CTB interaction. This indicates that several other amino acids found in the CDR regions contribute with steric effects, as opposed to direct epitope-paratope binding interactions, and can be exchanged for amino acids with similar properties (e.g., size and charge). For example, a Tryptophan found in CDR2 (W59) could be exchanged for another amino acid with a hydrophobic side change and similar size such as Tyrosine (W59Y). Similarly, an Asparagine found in CDR3 (N108) could be exchanged for the similar Serine (N108S). One can expect that these types of mutations (e.g., W59Y and N108S) probably keep the binding affinity of BP-L towards CTB and maintain a similar CTB-GM1 blocking capacity.

[0139] Conclusion

[0140] Based on these findings, it is evident that the skilled person may use the new BP-L sequence information herein in combination with open access protein structure prediction tools (e.g., ColabFold) to make minor changes in e.g., the CDR regions, while maintaining herein relevant CTB-GM1 blocking capacity.

[0141] REFERENCE LIST

[0142] 1 : Harmsen et al. (Appl Microbiol Biotechnol (2009) 84:1087-1094)

[0143] 2: WO2020 / 144164A1 (Bactolife)

[0144] 3: Ahmadi et al. (Sci Rep (2020) Jul 1 ;10(1):10765)

[0145] 4: Taheri et al. (International Immunopharmacology 89 (2020) 107054)

[0146] 5: Barati et al. (Iran J Immunol. 2018; 15(1):47-58)

[0147] 6: Garcia et al. (Scientific Reports, vol. 13, no. 1 , 15 March 2023)

[0148] 7: Legler et al. (Acta Cryst. (2013). F69, 90-93)

Claims

CLAIMS1 . A single-domain antibody (sdAb) which binds to subunit B of Vibrio cholerae toxin (CT), wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; for use in the reduction of risk, prevention or treatment of Vibrio cholerae infection in a subject.

2. The single-domain antibody for use of claim 1 , wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein 1 amino acid has been altered; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein 1 amino acid has been altered; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein 1 amino acid has been altered.

3. The single-domain antibody for use of claim 2, wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3.

4. The single-domain antibody for use of any of the preceding claims, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 90% sequence identity thereto.

5. The single-domain antibody for use of any of the preceding claims, wherein the single-domain antibody is a fusion protein comprising a single-domain antibody as defined in any one of the preceding claims and one or more further single-domain antibodies as defined in any one of the preceding claims, and optionally one or more linkers, such as one or more GS linkers.

6. The single-domain antibody for use of claim 5, wherein the fusion protein is a homodimer or a heterodimer.

7. The single-domain antibody for use of claim 6, wherein the fusion protein is a homodimer.

8. The single-domain antibody for use of claim 7, wherein the fusion protein homodimer is comprising a single-domain antibody as defined in claim 3 and one more single-domain antibody as defined in claim 3.

9. The single-domain antibody for use of claims 7 or 8, wherein the fusion protein homodimer is comprising or consisting of the sequence as set forth in SEQ ID NO: 5, or a sequence having at least 90% sequence identity thereto.

10. The single-domain antibody for use of claim 9, wherein the fusion protein homodimer is comprising or consisting of the sequence as set forth in SEQ ID NO: 5.

11. The single-domain antibody for use of any of the preceding claims, wherein the single-domain antibody also binds to heat-labile enterotoxin (LT) secreted by enterotoxigenic Escherichia coli (ETEC) and wherein the single-domain antibody is also for use in the reduction of risk, prevention or treatment of enterotoxigenic Escherichia coli (ETEC) infection in a subject.

12. The single-domain antibody for use of any of the preceding claims, wherein the single-domain antibody is a glycosylated single-domain antibody.

13. The single-domain antibody for use of claim 12, wherein the glycosylated single-domain antibody had been obtained by recombinant expression in Aspergillus oryzae.

14. The single-domain antibody for use of claim 13, wherein the glycosylated single-domain antibody is a fusion protein homodimer antibody of any of claims 8-10.

15. The single-domain antibody for use of any of the preceding claims, wherein the subject is a human.

16. The single-domain antibody for use of any of the preceding claims, wherein the single-domain antibody (sdAb) is capable of blocking the CT-GM1 receptor interaction with a blocking capacity of at least 50% (preferably at least 70%, more preferably at least 90%, and most preferably at least 98%) at sdAb:CTB binding site ratio of 2:1 , measured according to working Example 1 herein.

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