Improved immunoglobulin single variable domains that inhibit urease and use thereof

Abstract

The present invention relates to polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, which ISVD is modified to improve one or more of its protease resistance, thermostability, producibility in microbial hosts and in situ performance in animal excreta. Alone or in combination, the ISVD's of the invention are able to inhibit a broad range of microbial and plant-based ureases. The urease-inhibiting polypeptides of the invention can be used in compositions such as animal feed, animal feed premixes and supplements, to be fed to animals, for reducing the amount of ammonia released from the animal and / or for reducing the amount of ammonia released from the animal's excreta, with the aim to reduce ammonia emissions from agriculture into atmosphere and / or ecosystems. In addition, in the polypeptides can be used in an environmental application like stable floors, slurry pits and urea-based fertilizers for reducing the emission of ammonia.

Description

[0001] Improved immunoglobulin single variable domains that inhibit urease and use thereof

[0002] Field of the invention

[0003] The present invention relates to the field of immunology, enzymology, agriculture and animal husbandry. In particular, the invention relates to improved polypeptides comprising immunoglobulin single variable domains (ISVDs) that inhibit the enzymatic activity of ureases, which polypeptide comprise modification in their amino acid sequences that improve properties such as protease resistance. The invention further relates the use of such improved polypeptides for reducing ammonia release from animals and from animal excreta in e.g. the stables and slurry pit.

[0004] Background of the invention

[0005] Ammonia is a highly reactive, pungent gas formed of nitrogen and hydrogen. Ammonia occurs in essential biological processes and is not a problem in low concentrations. However, ammonia emissions into the atmosphere have negative consequences for ecosystems and human and animal health.

[0006] Ammonia reacts with air humidity to form ammonium (NH4). Ammonium depositions contribute to acidification of land and water. Deposition of ammonium degrades the biochemistry of natural ecosystems and causes eutrophication. Ammonia combines with other air pollutants such as sulfuric acid and nitric acid to form secondary particulate matter (PM10), which stays in the air over several days and travels long distances. Particulate matter contributes to respiratory diseases.

[0007] Ammonia pollution from agriculture represents a high cost to society. According to the European Nitrogen Assessment, it is estimated at 12 € per kg of emitted nitrogen for health damages an 2 € for ecosystem damages (Brink C, van Grinsven H, 2011 : Cost and benefits of nitrogen in the environment. The European Nitrogen Assessment, chapter 22, Cambridge University Press).

[0008] Livestock excreta contain high amounts of ammonia. They are at the origin of 75 % of all ammonia emissions from agriculture in the EU and therefore need to be minimized. Upon digestion of nitrogen containing materials, in the animals’ digestive system urea may be broken down and converted into ammonia by plant- and / or microbial-derived ureases. In addition, urea expelled in the urine may be converted to ammonia on the ground by contact with plant- and / or microbial- derived ureases present in the feces or soil.

[0009] Urease inhibitors such as N-(n-butyl) thiophosphoric triamide (NBPT) are widely used to reduce ammonia volatilization from the use of urea as nitrogen fertilizer for farmlands (see e.g. Cantarella et al., 2018, J. Adv. Res. 13: 19-27). However, due to their suspected toxicity such inhibitors are less suitable for use in animals.

[0010] WO 2021 / 258059 discloses the use of saponin as additive to animal feed for inhibiting the release of gaseous ammonia through the inhibition of urease activity.

[0011] Hoseinpoor et al. (Appl Biochem Biotechnol. 2014, 172:3079-3091) describe camel heavychain antibodies against the UreC subunit of urease from Helicobacter pylori for use in the treatment of H. pylori infection. CN 109206519 describe camel heavy-chain antibodies against the B subunit of urease from

[0012] Helicobacter pylori for use in the treatment of H. pylori infection.

[0013] Co-pending application PCT / EP2024 / 053281 discloses immunoglobulin single variable domains (ISVDs), in particular VHHs, that bind to and inhibit plant and / or bacterial ureases, amongst others for use in reducing ammonia release from animals and from animal excreta in e.g. the stables and slurry pit. There is however, still a need for such urease-inhibiting ISVDs domains that are improved in terms of one or more of protease resistance, thermostability, producibility in microbial hosts and in situ performance in animal excreta.

[0014] It is an object of the present invention to provide for such improved urease-inhibiting ISVDs for use in reducing ammonia release from animals and / or from animal excreta.

[0015] Summary of the invention

[0016] In a first aspect, there is provided a polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein: a) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, A15P, R90Q, A96P, D97E, S100A and A54C - I78C, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 10; b) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the ISVD comprises at least one modification selected from the group consisting of R28F, A54C - I78C, Q1 E, Q5V and A15P, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 3; or, c) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 7.

[0017] In an embodiment, there is provided a polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein a) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99 sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises the following mutations: Q1 E, Q5V, R90Q, A96P and S100A, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 10; b) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the ISVD comprises the following mutations: Q1 E, Q5V and R28F and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 3; or, c) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises the following mutations: Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 7.

[0018] In an embodiment, there is provided a polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein a) the ISVD comprises an amino acid sequence having SEQ ID NO: 22, or having at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 22, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 22; b) the ISVD comprises an amino acid sequence having SEQ ID NO: 23, or having at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 23, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 23; or, c) the ISVD comprises an amino acid sequence having SEQ ID NO: 21 , or having at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 .

[0019] In one embodiment, the polypeptide is a polypeptide wherein: a) the ISVD comprising CDRs as set forth in SEQ ID NO: 10, comprises a modification or a combination of modifications selected from the group consisting of: i) A54C - I78C; ii) Q1 E, Q5V and A15P; iii) Q1 E and Q5V; iv) R90Q, A96P, D97E and S100A; v) Q5V, R90Q, A96P, D97E and S100A; vi) Q1 E, Q5V, R90Q, A96P, D97E and S100A; vii) Q1 E, Q5V, R90Q, A96P and D97E; viii) Q1 E, Q5V, R90Q, A96P and S100A; ix) Q1 E, Q5V, R90Q, D97E and S100A; x) Q1 E, Q5V, A96P, D97E and S100A; xi) Q1 E, Q5V, R90Q and A96P; xii) Q1 E, Q5V, R90Q and D97E; xiii) Q1 E, Q5V, R90Q and S100A; xiv) Q1 E, Q5V, A96P and D97E; xv) Q1 E, Q5V, A96P and S100A; xvi) Q1 E, Q5V, D97E and S100A; xvii) Q1 E, Q5V and R90Q; xviii) Q1 E, Q5V and A96P; xix) Q1 E, Q5V and D97E; xx) Q1 E, Q5V and S100A; and, xxi) Q1 E, Q5V, R90Q, A96P, D97E, S100A and A54C - I78C; b) the ISVD comprising CDRs as set forth in SEQ ID NO: 3, comprises a modification or a combination of modifications selected from the group consisting of: i) R28F; ii) A54C - I78C; iii) R28F and A54C - I78C; iv) Q1 E, Q5V and A15P; v) Q1 E and Q5V; vi) Q1 E, Q5V and R28F; and, vii) Q1 E, Q5V, R28F and A54C - I78C; or, c) the ISVD comprising CDRs as set forth in SEQ ID NO: 7, comprises a modification or a combination of modifications selected from the group consisting of: i) Q1 E and Q5V; ii) K84T; iii) R108Q; iv) A54C - I78C; v) Q1 E, Q5V and K84T; vi) Q1 E, Q5V and R108Q; vii) Q1 E, Q5V and A54C - I78C; viii) Q1 E, Q5V, K84T and R108Q; ix) Q1 E, Q5V, K84T and A54C - I78C; x) Q1 E, Q5V, R108Q and A54C - I78C; xi) K84T, R108Q and A54C - I78C; and xii) Q1 E, Q5V, K84T, R108Q and A54C - I78C.

[0020] In one embodiment, the polypeptide is a polypeptide wherein: a) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 10, except for the at least one modification or combination of modifications defined in a) above; b) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 3, except for the at least one modification or combination of modifications defined in b) above; or, c) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 7, except for the at least one modification or combination of modifications defined in c) above.

[0021] In one embodiment, the polypeptide is a polypeptide wherein: a) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 22; b) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 23 or, c) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 21.

[0022] In one embodiment, the polypeptide is a polypeptide wherein the CDRs are Kabat CDRs, Chothia CDRs or IMGT CDRs.

[0023] In one embodiment, the polypeptide comprises more than one ISVD as defined above, wherein, optionally the ISVDs are linked through a spacer amino acid sequence, wherein preferably the polypeptide comprising at least two different ISVDs.

[0024] In one embodiment, the polypeptide comprises: a) a first ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, and a second ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3; b) a first ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, and a second ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7; c) a first ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, and a second ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7; or, d) a first ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, a second ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, and a third ISVD that is an ISVD as defined above, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7.

[0025] In a second aspect, there is provided a composition comprising a polypeptide as defined above, wherein preferably, the composition is suitable for feeding or administering to a non-human animal. In one embodiment, the composition comprises more than one different polypeptides as defined herein. In one embodiment, the composition comprises a polypeptide as defined in the first aspect, in combination with a polypeptide comprising an ISVD that comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in a sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23, wherein preferably, the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in the sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23, more preferably, the ISVD comprises an amino acid sequence as set forth in the sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23 more preferably, the ISVD comprises an amino acid sequence as set forth in the sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23.

[0026] In a third aspect, there is provided a nucleic acid encoding a polypeptide as defined in the first aspect. Fourth, fifth and sixth aspects are described herein below in more detail.

[0027] In a seventh aspect, there is provided a method for inhibiting urease activity in the gastrointestinal tract of an animal, wherein the method comprises feeding or administering to the animal a polypeptide as defined in the first aspect or a composition as defined in the second aspect.

[0028] In an eighth aspect, there is provide a method for reducing at least one of the amount of ammonia released from an animal and the amount of ammonia released from the animal excreta, wherein the method comprises feeding or administering to the animal a polypeptide as defined in the first aspect or a composition as defined in the second aspect.

[0029] In a nineth aspect, there is provided a use of a polypeptide as defined in the first aspect or a composition as defined in the second aspect, for at least one of: a) reducing the amount of ammonia released from an animal, wherein, preferably the amount of ammonia released from an animal is reduced by inhibiting urease activity in the gastro-intestinal tract of the animal; b) reducing the amount of ammonia released from the excreta of animal, wherein, preferably the amount of ammonia released from the animal’s excreta is reduced by inhibiting urease activity in the animal’s excreta, more preferably in the feces of the animal; c) reducing the amount of ammonia released from animal feeding operations; preferably for reducing the amount of ammonia released from feeding operations into the atmosphere; d) preventing the loss of nitrogen-value in manure; e) reducing ammonia volatilization from the use of urea as nitrogen fertilizer; and, f) reducing the amount of ammonia released from a surface comprising urease from microbial or vegetal sources, for example by applying the composition or polypeptide onto the surface.

[0030] In a tenth aspect, there is provided a use of a polypeptide as defined in the first aspect or a composition as defined in the second aspect, for reducing the amount of ammonia released from an animal’s excreta by applying a composition comprising the polypeptide onto the animal’s excreta.

[0031] In an eleventh aspect, there is provided a polypeptide as defined in the first aspect or a composition as defined in the second aspect, for use in the prevention or treatment of hyperammonemia. Description of the invention

[0032] Definitions

[0033] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0034] “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0035] “About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0036] “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0037] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0038] Exemplary": this term means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

[0039] As used herein, "in combination with" is intended to refer to all forms of administration that provide a first agent together with a further (second, third) agent. The agents may be administered simultaneous, separate or sequential and in any order. Agents administered in combination have biological activity in the animal to which the agents are delivered.

[0040] “Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The terms “sequence identity” or “sequence similarity” means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over the entire length (of at least the shortest sequence in the comparison) and maximizing the number of matches and minimizes the number of gaps such as by the programs ClustalW (1.83), GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) I 8 (proteins) and gap extension penalty = 3 (nucleotides) I 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences of the invention is ClustalW (1 .83) using a blosum matrix and default settings (Gap opening penalty:10; Gap extension penalty: 0.05). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

[0041] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0042] Alternative conservative amino acid residue substitution classes.

[0043] Alternative physical and functional classifications of amino acid residues.

[0044] The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be a compound or a composition. An agent can e.g. be selected from the group consisting of: polynucleotides, polypeptides, small molecules, (multispecific) antigen binding proteins, such as antibodies and functional fragments thereof.

[0045] Unless indicated otherwise, the terms "immunoglobulin” and “antibody" whether it used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more specific interpretation. The term "antigen-binding domain" or "antigen-binding region" refers to the portion of an antigen-binding protein that is capable of specifically binding to an antigen or epitope. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region, e.g. comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding domain is an immunoglobulin-derived antigen-binding domain from a single domain antibody consisting only of heavy chains and devoid of light chains as are known e.g. from camelids, wherein the antigen-binding site is present on, and formed by, the single variable domain (also referred to as an "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include the single variable domains of camelid heavy chain antibodies (VHH, also denoted as VHH), also known as nanobodies, domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, an antigen-binding domain comprises a non- immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as DARPpins; Affilins; anticalins, etc.

[0046] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH", or to “VHH” in case of a heavy chain antibody such as the camelid antibodies that consist of only heavy chains. The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the average 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (HVRs) or complementarity determining regions (CDRs) that are usually each about 9-12 amino acids long, although the CDR3 of VHHs can be much longer, e.g. 18 amino acids or more. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a p-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the p-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0047] The terms “VHH”, “VHH domain” and “nanobody” are interchangeable herein and are used herein to refer to the variable domain of a heavy chain antibody, i.e. an antibody consisting only of heavy chains and devoid of light chains as are known e.g. from camelids. The amino acid sequence and structure of a VHH can be considered without however being limited thereto to be comprised of four framework regions or "FR's", which are referred to in the art and herein below as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or"CDRs", which are referred to in the art as "Complementarity Determining Region 1" or"CDR1"; as "Complementarity Determining Region 2" or"CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. The total number of amino acid residues in a VHH can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments or analogs (as further described herein below) of a VHH are not particularly limited as to their length and / or size, as long as such parts, fragments or analogs meet the further functional requirements outlined herein below and are also preferably suitable for the purposes described herein.

[0048] The amino acid residues of a VHH (or conventional variable domain) are numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md. , Publication No. 91), as applied to VHH domains from Camelids by Riechmann and Muyldermans (1999, J. Immunol. Methods; 231 : 25-38; see for example Fig. 2 of said reference). According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36- 49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that as is well known in the art for VH domains and for VHH domains the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDRs, position 1 according to the Kabat numbering corresponds to the start of FR1 and visa versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and visa versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and visa versa, and position 103 according to the Kabat numbering corresponds to the start of FR4.

[0049] Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains from Camelids, are the method described by Chothia et al. (1989, Nature 342, 877-883), the so-called "AbM definition" and the so- called "contact definition". However, in the present description, claims and figures, the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0050] Table A. CDR defintions1

[0051] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0052] With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1 , a-CDRL2, a-CDR-L3, a-CDR-H1 , a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1 , 50-55 of L2, 89-96 of L3, 31- 35B of H1 , 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619- 1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to IMGT (Lefranc et al., supra).

[0053] For a general description of heavy chain antibodies and the variable VHH domains thereof, reference is inter alia made to the following references, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079, WO 96 / 34103, WO 94 / 25591 , WO 99 / 37681 , WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301 , EP 1134231 , WO 02 / 48193, WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016, WO 03 / 055527 WO 03 / 050531 , WO 01 / 90190, WO 03 / 025020; WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863 and WO 04 / 062551 and Hassanzadeh-Ghassabeh et al. (2013, Nanomedicine, 8(6):1013-1026). For a more specific description of single domain VHH antibodies against Von Willebrand Factor or platelet receptor GPIb, reference is made to WO 2004 / 062551 and WO 2006 / 122825.

[0054] Generally, it should be noted that the term “VHH” (or nanobody) as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, VHHs as used in the invention can be obtained (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "camelization" of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (4) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (5) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and / or (6) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing are state of the art and therefore known to the skilled person.

[0055] The term "valent" or "valency" as used within the current application denotes the presence of a specified number of binding sites in an antigen binding molecule. As such, the terms "bivalent", "tetravalent", and "hexavalent" denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule.

[0056] As used herein, the term "affinity matured" in the context of antigen binding molecules (e.g., antibodies) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, e.g., by mutation, binds to the same antigen, preferably binds to the same epitope, as the reference antibody; and has a higher affinity for the antigen than that of the reference antigenbinding molecule. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0057] The term "specifically binds" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigenbinding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Affinity can be determined in a manner known per se, depending on the specific combination of antigen-binding protein and antigen of interest. Avidity is herein understood to refer to the strength of binding of a target molecule with multiple binding sites by a larger complex of binding agents, i.e. the strength of binding of multivalent binding. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding protein and the valency, i.e. the number of binding sites present on the antigen-binding protein. Affinity, on the other hand refers to simple monovalent receptor ligand systems.

[0058] Typically, an antigen-binding region of a multispecific antigen binding protein of the invention thereof will specifically bind its target molecule (antigen) with a dissociation constant (KD) of about 10'6to 10'12M or less, and preferably 10-8to 10-12M or less, and / or with a binding affinity of at least 10-6M or 10-7M, preferably at least 10-8M, more preferably at least 10-9M, such as at least 1 O-10, 10’11, 10-12M or more. Any KD value greater than 10-4M (i.e. less than 100 pM) is generally considered to indicate non-specific binding. Thus, an antigen-binding region that “specifically binds” an antigen, is an antigen-binding domain that binds the antigen with a KD value of no more than 1 O’

[0059] 4M, as may be determined as herein described below. Preferably, an antigen-binding region of a multispecific antigen binding protein of the invention will specifically bind to the target molecule with an affinity less than 800, 400, 200, 100, 50, 20, 10 or 5 nM, more preferably less than 1 nM, such as less than 500, 200, 100, 50, 20, 10 or 5 pM. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention (see e.g. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds.. Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983)). Specific illustrative embodiments are described in the following.

[0060] A "Kd" or "Kd value" can be measured by using an ELISA as known in the art or by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™- 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 - 50 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl- N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, into

[0061] 5 pg / ml (~0.2 pM) before injection at a flow rate of 5pl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of the antibody or Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25pl / min. Association rates (kon) and dissociation rates (kotr) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865- 881 . If the on-rate exceeds 106M-1S’1by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

[0062] A “nucleic acid construct” or “nucleic acid vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term “nucleic acid construct” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms “expression vector” or expression construct" refer to nucleic acid molecules that are capable of effecting expression of a nucleotide sequence or gene in host cells or host organisms compatible with such expression vectors or constructs. These expression vectors typically include regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed to effect its expression. Such regulatory elements usually at least include suitable transcription regulatory sequences and optionally, 3’ transcription termination signals. Additional elements necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention whereas an expression construct will usually integrate in the host cell’s genome for it to be maintained. Techniques for the introduction of nucleic acid into cells are well established in the art and any suitable technique may be employed, in accordance with the particular circumstances. The introduced nucleic acid may be on an extra- chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques. The introduction may be followed by expression of the nucleic acid to produce the encoded fusion protein. In some embodiments, host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded fusion protein polypeptide is produced, when an inducible promoter is used, expression may require the activation of the inducible promoter.

[0063] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.

[0064] The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term “reporter” may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional.

[0065] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

[0066] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin.

[0067] Amino acid substitutions are herein indicated as AOXAS, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence, and As indicates the substitute amino acid as present in that position in the modified amino acid sequence. For example, I8H denotes that the original amino acid isoleucine (I) in position 8 is changed to a histidine (H). A combination of two substitutions that introduces additional disulphide bond is indicated as A01X1C - A02X2C, wherein A01 and A02 indicate the original amino acids at positions Xi and X2, respectively, that are replaced by cysteine residues.

[0068] Amino acid deletions are herein indicated as A0X-, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence and the dash indicates that the original amino acid Aois no longer present in the modified amino acid sequence. For example N59- indicates that the asparagine (N) in position 59 is deleted in the modified amino acid sequence.

[0069] Amino acid insertion are herein indicated as A0X insAi-n, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence and insAi-n indicates that amino acids 1 - n replace the original amino acid Ao. For example G84 insGGGGG indicates that the original glycine in position 84 is replaced by a sequence of 5 glycines in the modified amino acid sequence.

[0070] The term “signal peptide” (sometimes referred to as signal sequence) is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by signal peptidase either during or after completion of translocation (from the cytosol into the secretory pathway, i.e. ER) to generate a free signal peptide and a mature protein. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species however the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in the art e.g. from Kall et al. (2004 J. Mol. Biol. 338: 1027- 1036) and von Heijne (1985, J Mol Biol. 184 (1): 99-105).

[0071] The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site. “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

[0072] The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and / or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

[0073] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

[0074] Any reference to nucleotide or amino acid sequences accessible in public sequence databases herein refers to the version of the sequence entry as available on the filing date of this document.

[0075] Detailed description of the invention

[0076] The present invention relates to improvements in polypeptides that inhibit bacterial and / or plant urease activity for use in reducing ammonia emissions from animal feeding operations. The polypeptides with urease inhibiting activity have been modified to improve one or more of their protease resistance, thermostability, producibility in microbial hosts and in situ performance in animal excreta. The improved polypeptides with urease inhibiting activity can be administered to the animal in any way, preferably as part of the animal diet, e.g. by feeding the animal a feed comprising the polypeptide with urease inhibiting activity. Alternatively, the improved polypeptides with urease inhibiting activity can be applied (ex vivo) to the animal’s excreta to prevent and / or reduce ammonia release therefrom. In another embodiment, the improved polypeptides with urease inhibiting activity can be applied (ex vivo) to nitrogen fertilizers (comprising urea) to reduce the rate of ammonia release therefrom.

[0077] In a first aspect therefore, there is provided a polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits an urease.

[0078] The term “urease” as used herein is an amidohydrolase according to enzyme classification EC 3.5.1.5 that catalyzes the hydrolysis of urea into carbon dioxide and ammonia: (NH2)2CO + H2O urease —> CO2 + 2NH3. Urease activity and its inhibition by a polypeptide as described herein can be assayed essentially as described in co-pending application PCT / EP2024 / 053281 .

[0079] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least one of a plant urease and a microbial urease. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least one of a plant urease and a bacterial urease. A plant urease is understood as a urease that naturally occurs in a plant. Likewise, a microbial urease is understood as a urease that naturally occurs in a microorganism and a bacterial urease is understood as a urease that naturally occurs in a bacterium.

[0080] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease. The polypeptide as described herein preferably comprises an antigen-binding domain that inhibits a plant urease of a plant species or cultivar that is commonly used as, or as an ingredient of animal feed. Such plants include species and / or cultivars of grass, sorghum, wheat, oats, barley, rice, corn, and legumes such as soybeans and Jack beans. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a leguminous urease, preferably the antigen-binding domain inhibits at least one of a Jack bean (Canavalia ensiformis) urease and a soy (Glycine max) urease.

[0081] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a microbial urease. The polypeptide as described herein preferably comprises an antigen-binding domain that inhibits a microbial urease of a microorganism that commonly occurs in the digestive tracts of animals, e.g., in the digestive tract of a ruminant. Such microorganisms in bacteria as mentioned below but also fungi such as anaerobic gut fungi e.g. of the class Neocallimastigomycetes, including fungi of the genera Anaeromyces, Caecomyces, Neocallimastix, and Piromyces.

[0082] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a bacterial urease. The polypeptide as described herein preferably comprises an antigen-binding domain that inhibits a bacterial urease of a bacterium that commonly occurs in the digestive tracts of animals, e.g., in the digestive tract of a ruminant. Such bacteria include species of the genera Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, Lactobacillus, Proteus, Providencia and Morganella. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a Klebsiella aerogenes urease, preferably the antigenbinding domain inhibits at least a Klebsiella aerogenes urease.

[0083] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least both a plant urease and a bacterial urease, preferably a plant urease and a bacterial urease as defined above. Different plant species, as well as different microbial species produce ureases, which differ from each other in their amino acid sequences. Most urease-inhibiting VHHs are species specific in that they inhibit only a limited spectrum of ureases from either plant or bacterial sources. However, in order to reduce ammonia emission from farm-animals and / or in the environment, a polypeptide as described herein preferably comprises an antigen-binding domain that inhibits the activity of a wide spectrum of ureases from the different plant or microbial sources. Alternatively, wide spectrum inhibitory activity can be obtained by combining more than one different urease-inhibiting antigen-binding domains with complementary inhibitory spectra into one polypeptide, or into one composition.

[0084] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits activity of a plant urease and that does not specifically bind to a urease from Helicobacter pylori. In one embodiment, a polypeptide as described herein comprises an antigenbinding domain that inhibits activity of a microbial urease and that does not specifically bind to a urease from Helicobacter pylori. A urease-inhibiting polypeptide for the treatment of H. pylori preferably inhibits a urease from H. pylori, but does not inhibit the activity of other (microbial or plant) ureases, i.e. the polypeptide is specific for the inhibition of H. pylori ureases. In contrast, the polypeptides as defined herein are preferably wide spectrum inhibitors, meaning that the inhibiting activity of the polypeptide is not limited to any specific urease. Instead the polypeptides as described herein can preferably inhibit the activity of several (microbial and / or plant) ureases prevalent in the gut.

[0085] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 80%, 90%, 99% or 100% activity of at least one of a plant urease and a bacterial urease as defined above, when assayed at a twofold molar excess of the antigen-binding domain. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 80%, 90%, 99% or 100% activity of a plant urease as defined above, when assayed at a twofold molar excess of the antigen-binding domain. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 80%, 90%, 99% or 100% activity of a microbial urease as defined above, when assayed at a twofold molar excess of the antigen-binding domain.

[0086] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an affinity for at least one of a plant urease and a bacterial urease as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an affinity for a plant urease as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an affinity for a bacterial urease as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM.

[0087] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits a urease as defined above in a competitive manner, as may be determined by enzyme inhibition kinetics using methods generally known in the art.

[0088] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an inhibitory constant (Ki) for inhibition of at least one of a plant and a bacterial urease as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an inhibitory constant (Ki) for inhibition of a plant as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an inhibitory constant (Ki) for inhibition of a bacterial urease as defined above, that is less than 800, or with increasing preference 400, 200, 100, 50, 20, 10 or 5 nM, preferably less than 1 nM, more preferably less than 500, or with increasing preference 200, 100, 50, 20, 10 or 5 pM.

[0089] An inhibitory constant for inhibition of a plant and / or a bacterial urease is herein defined as the concentration of the antigen-binding domain that is required in order to decrease the maximal rate of the urease reaction by half, i.e., the concentration required to produce half maximum inhibition. The inhibitory constant of an antigen-binding domain for inhibition of a urease may be determined by enzyme inhibition kinetics using methods generally known in the art.

[0090] In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an IC50 for inhibition of at least one of a plant and a bacterial urease as defined above, that is less than 30, or with increasing preference 10, 5, 4, 3, 2, 1.0, 0.7, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml, preferably less than 2, or with increasing preference 1.0, 0.7, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml more preferably less than 0.5, or with increasing preference 0.4, 0.3, 0.25, 0.2, or 0.1 1 pg / ml. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an IC50 for inhibition of a plant urease as defined above, preferably a Jack bean urease that is less than 2, or with increasing preference 1.0, 0.7, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml, preferably less than 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml more preferably less than 0.3, or with increasing preference 0.25, 0.2, or 0.11 pg / ml. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that has an IC50 for inhibition of a bacterial urease as defined above, that is less than 30, 10, 5, 4, 3, 2, 1 .0, 0.7, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml, preferably less than 2, 1 .0, 0.7, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml more preferably less than 0.5, 0.4, 0.3, 0.25, 0.2, or 0.11 pg / ml. An IC50 constant for inhibition of a plant and / or a bacterial urease is herein defined as the concentration of the antigen-binding domain that is required in order to decrease the maximal rate ofthe urease reaction by half, i.e., the concentration required to produce half maximum inhibition. The inhibitory constant of an antigen-binding domain for inhibition of a urease may be determined by enzyme inhibition kinetics using methods generally known in the art, for example as described in as described in co-pending application PCT / EP2024 / 053281 .

[0091] In one embodiment, a polypeptide as described herein comprises an anti-urease antigenbinding domain that is resistant to proteases. For inhibition of urease activity in the gastrointestinal tract after oral application of a polypeptide as described herein should be able to resist proteolytic degradation. In one embodiment, a polypeptide as described herein comprises an anti-urease antigen-binding domain that is resistant to at least one protease in the gastrointestinal tract, preferably at least one of pepsin, trypsin and chymotrypsin, more preferably at least one of bovine pepsin, trypsin and chymotrypsin. Thus, in one embodiment, there is provided a polypeptide as described herein, wherein the polypeptide is protease resistant in that after a preincubation for 1 hour at 37 °C of the polypeptide at 0.1 mg / ml, with at least one of pepsin at 0.001 mg / ml, trypsin at 0.1 mg / ml and chymotrypsin at 0.1 mg / l, under condition described in the examples (and / or in Harmsen et al., 2006), at least 10%, at least 25%, at least 50%, or at least 75% of the polypeptide functionally binds the antigen of the antigen-binding domain comprised in the polypeptide, as determined in an ELISA assay, preferably an ELISA assay as described in the examples (and / or in Harmsen et al., 2006).

[0092] In one embodiment, the antigen-binding domain comprised in a polypeptide as described herein is an immunoglobulin single variable domain (ISVD). In a preferred embodiment, the ISVD comprised in a polypeptide as described herein is a single variable domains of camelid heavy chain antibodies, i.e., a VHH. Preferably the VHH is a VHH of the genus Lama, more preferably the VHH is a VHH of the species Lama glama.

[0093] In one embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in one of SEQ ID NO.’s: 1 - 17, wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO.’s: 1 - 17, respectively, and wherein the amino acid sequence comprises at least one modification, wherein preferably, the amino acid sequence comprises at least one modification as compared to the amino acid sequence of SEQ ID NO.’s: 1 - 17, respectively. It is understood here that the at least one modification can be a substitution, deletion and / or insertion of at least one amino acid. In one embodiment, a polypeptide as described herein comprises at least one amino acid modification selected from the group consisting of: Q1 E, Q5V, A15P, R28F, K84T, R90Q, A96P, D97E, S100A, R108Q and A54C - I78C. In one embodiment, a polypeptide as described herein comprises at least two, three, four, five, six, seven or eight amino acid modifications selected from the group consisting of: Q1 E, Q5V, A15P, R28F, K84T, R90Q, A96P, D97E, S100A, R108Q and A54C - I78C. In one embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, A15P, R90Q, A96P, D97E, S100A and A54C - I78C, and wherein the ISVD comprises CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10. In a preferred embodiment, the ISVD at least comprises a modification or a combination of modifications selected from the group consisting of: i) A54C - I78C; ii) Q1 E, Q5V and A15P; iii) Q1 E and Q5V; iv) R90Q, A96P, D97E and S100A; v) Q5V, R90Q, A96P, D97E and S100A; vi) Q1 E, Q5V, R90Q, A96P, D97E and S100A; vii) Q1 E, Q5V, R90Q, A96P and D97E; viii) Q1 E, Q5V, R90Q, A96P and S100A; ix) Q1 E, Q5V, R90Q, D97E and S100A; x) Q1 E, Q5V, A96P, D97E and S100A; xi) Q1 E, Q5V, R90Q and A96P; xii) Q1 E, Q5V, R90Q and D97E; xiii) Q1 E, Q5V, R90Q and S100A; xiv) Q1 E, Q5V, A96P and D97E; xv) Q1 E, Q5V, A96P and S100A; xvi) Q1 E, Q5V, D97E and S100A; xvii) Q1 E, Q5V and R90Q; xviii) Q1 E, Q5V and A96P; xix) Q1 E, Q5V and D97E; xx) Q1 E, Q5V and S100A; and, xxi) Q1 E, Q5V, R90Q, A96P, D97E, S100A and A54C - I78C. In an embodiment, a polypeptide as described herein comprises an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises the following mutations: Q1 E, Q5V, R90Q, A96P and S100A, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 10. In a more preferred embodiment, the ISVD comprises the CDRs as set forth in SEQ ID NO: 10. In a most preferred embodiment, the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 10, except for the above-mentioned at least one modification or combination of modifications.

[0094] In an embodiment, a polypeptide as described herein comprises an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 22, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 22. In a most preferred embodiment, the polypeptide is a polypeptide wherein the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 22.

[0095] In one embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the ISVD comprises at least one modification selected from the group consisting of R28F, A54C - I78C, Q1 E, Q5V and A15P, and wherein the ISVD comprises CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3. In a preferred embodiment, the ISVD at least comprises a modification or a combination of modifications selected from the group consisting of: i) R28F; ii) A54C - I78C; iii) R28F and A54C - I78C; iv) Q1 E, Q5V and A15P; v) Q1 E and Q5V; vi) Q1 E, Q5V and R28F; and, vii) Q1 E, Q5V, R28F and A54C - I78C.

[0096] In an embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the ISVD comprises the following mutations: Q1 E, Q5V and R28F and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 3. In a more preferred embodiment, the ISVD comprises the CDRs as set forth in SEQ ID NO: 3. In a most preferred embodiment, the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 3, except for the above-mentioned at least one modification or combination of modifications.

[0097] In an embodiment, a polypeptide as described herein comprises an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 23, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 23. In a most preferred embodiment, the polypeptide is a polypeptide wherein the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0098] In one embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7. In a preferred embodiment, the ISVD at least comprises a modification or a combination of modifications selected from the group consisting of: i) Q1 E and Q5V; ii) K84T; iii) R108Q; iv) A54C - I78C; v) Q1 E, Q5V and K84T; vi) Q1 E, Q5V and R108Q; vii) Q1 E, Q5V and A54C - I78C; viii) Q1 E, Q5V, K84T and R108Q; ix) Q1 E, Q5V, K84T and A54C - I78C; x) Q1 E, Q5V, R108Q and A54C - I78C; xi) K84T, R108Q and A54C - I78C; and xii) Q1 E, Q5V, K84T, R108Q and A54C - I78C. In an embodiment, a polypeptide as described herein comprises an ISVD that specifically binds and inhibits an urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises the following mutations: Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 7. In a more preferred embodiment, the ISVD comprises the CDRs as set forth in SEQ ID NO: 7. In a most preferred embodiment, the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 7, except for the above-mentioned at least one modification or combination of modifications.

[0099] In an embodiment, a polypeptide as described herein comprises an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 . In a most preferred embodiment, the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 21.

[0100] In one embodiment, in a polypeptide as described herein, the CDRs in the ISVD are Kabat CDRs, Chothia CDRs or IMGT CDRs. Kabat CDRs, Chothia CDRs and IMGT CDRs are herein understood to refer to CDRs the positions of which in an ISVD amino acid sequence are defined according to Kabat, Chothia or IMGT, as provided in Table A above.

[0101] In one embodiment, a polypeptide as described herein comprises more than one anti-urease ISVD as described herein, optionally linked through a spacer amino acid sequence. Thus, antigenbinding domain as described herein, e.g. an ISVD or VHH, can be in isolated form or essentially isolated form, or the antigen-binding domain can form part of a protein or polypeptide as described herein, which may comprise or essentially consist of one or more antigen-binding domains and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more antigen-binding domains may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other urease epitopes or relevant targets), so as to provide a monovalent, multivalent or multispecific polypeptide.

[0102] In a one embodiment, there is provided a bispecific polypeptide comprising a first ISVD as described herein, and a second ISVD as described herein, wherein the first and second ISVDs are different ISVDs, and wherein the first and second ISVDs are fused in a single polypeptide chain. In one embodiment of the bispecific polypeptide, the first and second polypeptides are linked through a spacer amino acid sequence, e.g. as defined below.

[0103] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above.

[0104] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above.

[0105] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above.

[0106] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 2, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above.

[0107] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 22, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above.

[0108] In one embodiment, the first ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above, and the second ISVD is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above.

[0109] In one embodiment, the first ISVD has CDRs as set forth in SEQ ID NO: 22, as defined above, and the second ISVD has CDRs as set forth in SEQ ID NO: 23, as defined above.

[0110] In one embodiment, the first ISVD has CDRs as set forth in SEQ ID NO: 22, as defined above, and the second ISVD has CDRs as set forth in SEQ ID NO: 21 , as defined above.

[0111] In one embodiment, the first ISVD is has CDRs as set forth in SEQ ID NO: 23, as defined above, and the second ISVD has CDRs as set forth in SEQ ID NO: 21 , as defined above.

[0112] In a one embodiment, there is provided a trispecific polypeptide comprising a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above, a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above, and a third ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above. In one embodiment of the trispecific polypeptide, the first, second and third polypeptides are linked through a spacer amino acid sequence, e.g. as defined below.

[0113] In a one embodiment, there is provided a trispecific polypeptide comprising a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 22, as defined above, a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above, and a third ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above. In one embodiment of the trispecific polypeptide, the first, second and third polypeptides are linked through a spacer amino acid sequence, e.g. as defined below. In a one embodiment, there is provided a trispecific polypeptide comprising a first ISVD that that has CDRs as set forth for the CDRs in SEQ ID NO: 22, as defined above, a second ISVD that has CDRs as set forth as set forth for the CDRs in SEQ ID NO: 23, as defined above, and a third ISVD that has CDRs as set forth as set forth for the CDRs in SEQ ID NO: 21 , as defined above. In one embodiment of the trispecific polypeptide, the first, second and third polypeptides are linked through a spacer amino acid sequence, e.g. as defined below.

[0114] When more than one antigen-binding domain is present in the polypeptide, the individual antigen-binding domains are preferably arranged in tandem, and preferably with suitable (flexible) spacer- or linker-amino acid sequences between the individual antigen-binding domains.

[0115] Suitable flexible linker-amino acid sequences are known in the art (e.g., from Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369). Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. Preferred flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of preferred (and widely used) flexible linker has one or more repeats of the sequence GGGGS (SEQ ID NO: 18), i.e. (Gly- Gly-Gly-Gly-Ser)n, wherein n can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility, such as e.g. the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 19) and EGKSSGSGSESKST (SEQ ID NO: 20), that have been applied for the construction of a bioactive scFv’s.

[0116] In a second aspect, there is provided a composition suitable for feeding or administering to a non-human animal comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease, as defined in the above first aspect.

[0117] Compositions for feeding non-human animals are well known in the art and include e.g. animal feed, animal feed premixes, animal feed supplements or animal drink compositions, such or drinking water or liquid feed composition. A composition a polypeptide comprising an ISVD that specifically binds and inhibits a urease can be any composition suitable for feeding or administering to a non-human animal. The animal feed supplement can also be a pharmaceutical composition comprising the polypeptide with urease inhibiting activity and a suitable carrier, which composition is administered to the animal in a manner known perse.

[0118] A non-human animal to which, orto whose excreta, a composition comprising the polypeptide with urease inhibiting activity is fed, administered and / or applied in accordance with the invention, can be any animal suitable for animal husbandry. Typically, such non-human animals include bovine, fowl, porcine, ovine, caprine, equine, and aquatic species. By way of example the non- human animals can include cattle, poultry, chickens, turkeys, ducks, quail, geese, pigs, goats, sheep, salmon, tilapia, trout and shrimp.

[0119] In one embodiment, the composition suitable for feeding or administering to a non-human animal is for (use in) reducing at least one of the amounts of ammonia released from the animal and ammonia released from the animal’s excreta. The polypeptide with urease inhibiting activity may thus exert its urease inhibiting activity in the animal’s digestive tract and / or after leaving the animal’s digestive tract, in the animal’s excreta. The term “excreta” is herein understood to include feces, urine as well as bird’s excrements and mixtures thereof.

[0120] In one embodiment, the composition comprises more than one different polypeptides as defined in the above first aspect. Thus in one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3, as defined above, a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, as defined above, and a third ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, as defined above.

[0121] In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 22, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 22, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above, and a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above. In one embodiment, the composition comprises a combination of a first ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 23, as defined above, a second ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 21 , as defined above, and a third ISVD that is a modified ISVD that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 22, as defined above.

[0122] In one embodiment, the composition comprises a combination of a first ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 22, as defined above, and a second ISVD that has CDRs as set forth forthe CDRs in SEQ ID NO: 23, as defined above. In one embodiment, the composition comprises a combination of a first ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 22, as defined above, and a second ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 21 , as defined above. In one embodiment, the composition comprises a combination of a first ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 23, as defined above, and a second ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 21 , as defined above. In one embodiment, the composition comprises a combination of a first ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 23, as defined above, a second ISVD that has CDRs as set forth for the CDRs in SEQ ID NO: 21 , as defined above, and a third ISVD that has CDRs as set forth in SEQ ID NO: 22, as defined above.

[0123] In a further embodiment, there is provided a composition comprising at least one polypeptide as defined in the above first aspect, in combination with a further polypeptide comprising an antiurease ISVD that inhibits the activity of at least one of a plant and a bacterial urease, such as described in co-pending application PCT / EP2024 / 053281 . Thus, in one embodiment, there is provided a composition comprising at least one polypeptide as defined in the above first aspect, in combination with a further polypeptide comprising an ISVD that comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in a sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23, wherein preferably the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in the sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23. In a preferred embodiment, the further polypeptide comprises an ISVD comprising an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23.

[0124] In one embodiment, a composition comprising a combination of more than one polypeptide as described herein and / or comprising a polypeptide as defined in the above first aspect, in combination with a further polypeptide comprising an anti-urease ISVD that inhibits the activity of at least one of a plant and a bacterial urease, such as described in co-pending application PCT / EP2024 / 053281 , inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 80%, 90%, 99% or 100% of the combined activities of a plant urease and a bacterial urease as defined above, when assayed at a twofold molar excess of the combined antigen-binding domains.

[0125] In a third aspect, there is provided a nucleic acid encoding a polypeptide comprising an ISVD that specifically binds and inhibits as described herein. Thus, the nucleic acid preferably is a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide. The nucleotide sequence encoding the polypeptide preferably encodes a signal peptide operably linked to the polypeptide. A nucleic acid molecule comprising the nucleotide sequence encoding the polypeptide, further preferably comprises regulatory elements for (or conducive to) the expression of the polypeptide in an appropriate host cell, which regulatory elements are operably linked to the nucleotide sequence. In one embodiment, the nucleotide sequence encoding the polypeptide comprises a codon-optimized coding sequence for a preferred host, such as Saccharomyces cerevisiae.

[0126] A fourth aspect relates to a host cell comprising the nucleic acid molecule comprising the nucleotide sequence encoding a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram-positive organisms, for example Escherichia coli or bacilli. Suitable yeast cells include Saccharomyces cerevisiae and Pichia pastoris. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (Gluzman et al., 1981 , Cell 23:175), L cells, HEK 293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI as described by McMahan et al., 1991 , EMBO J. 10: 2821 . Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). Host cells comprising the nucleic acid molecule of the invention can be cultured under conditions that promote expression of the polypeptide. Thus, a fifth aspect relates to a method for producing a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity as described herein, the method comprising the step of cultivating a host cell comprising the nucleotide sequence encoding the polypeptide, under conditions conducive to expression of the polypeptide, optionally, recovering the polypeptide. The polypeptide can be recovered by conventional protein purification procedures, including protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, using e.g. strepavidin / biotin (see e.g. Low et al., 2007, J. Chromatography B, 848:48-63; Shukla et al., 2007, J. Chromatography B, 848:28-39).

[0127] In a sixth aspect, there is provided a method for producing a composition suitable for feeding or administering to a non-human animal, an animal (drinking) water supplement or a pharmaceutical composition comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein. The method comprises at least the step of admixing the polypeptide with further ingredients of the composition suitable for feeding or administering to a non-human animal, or pharmaceutical composition.

[0128] A seventh aspect, relates to a method for inhibiting urease activity in the intestines or gastrointestinal tract of an animal, wherein the method comprises feeding the animal a composition comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove. The method preferably is not a medical and / or veterinary method of treating the human or animal body. Particularly, the method is not a method for preventing and / or treating a Helicobacter pylori infection.

[0129] In an eighth aspect, there is provided a method for reducing at least one of the amount of ammonia released from an animal and the amount of ammonia released from the animal excreta, wherein the method comprises feeding or administering to the animal a composition comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove. The composition fed or administered to the animal can be a composition suitable for feeding or administering to a non-human animal or a pharmaceutical composition as described above. The animal preferably is a non-human animal as herein defined. In one embodiment, the composition comprising the polypeptide at least partially passes though the digestive tract so that it is expelled in the feces, whereby the polypeptide may be available for inhibiting urease activity in the feces, particularly upon contact with urine urea, to provide for a decreased production of ammonia outside of the animal. In one embodiment, the composition comprising the polypeptide completely passes though the digestive tract so that it is entirely expelled in the feces. For this purpose, the composition may be provided with a coating that is sufficient to protect the urease inhibitor from digestion at least for a period of time sufficient for passage through the digestive tract. Such coatings are generally known in the art.

[0130] A nineth aspect relates to use of a composition comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for at least one of: a) reducing the amount of ammonia released from an animal, wherein, preferably the amount of ammonia released from an animal is reduced by inhibiting urease activity in the intestines or gastrointestinal tract of the animal; b) reducing the amount of ammonia released from the excreta of animal, wherein, preferably the amount of ammonia released from the animal’s excreta is reduced by inhibiting urease activity in the animal’s excreta, more preferably in the feces of the animal; c) reducing the amount of ammonia released from animal feeding operations; preferably for reducing the amount of ammonia released from feeding operations into the atmosphere; d) preventing the loss of nitrogen-value in manure; e) reducing ammonia volatilization from the use of urea as nitrogen fertilizer, e.g. for farmlands; f) reducing the amount of ammonia released from a surface comprising urease from microbial or vegetal sources, for example by applying the composition or polypeptide onto the surface; and, g) reducing the amount of ammonia formed in an animal’s intestines or gastrointestinal tract by inhibiting urease activity in the intestines or gastrointestinal tract of the animal, to improve the animal’s health and / or growth rate. In one embodiment, the composition comprising the polypeptide is a composition suitable for feeding or administering to a non-human animal, or a pharmaceutical composition as described above. The animal preferably is a non-human animal as herein defined.

[0131] In a tenth aspect, there is provided use of a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigenbinding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for reducing the amount of ammonia released from an animal’s excreta by applying a composition comprising the polypeptide onto the animal’s excreta. In one embodiment, a composition comprising the polypeptide is sprayed onto and / or mixed with the animal’s excreta, e.g., in stables and / or slurry pits. The animal preferably is a non-human animal as herein defined.

[0132] An eleventh aspect relates a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for use in the prevention and / or treatment of hyperammonemia. The polypeptide can be used for the prevention and / or treatment of hyperammonemia in a human subject or in a non- human animal, e.g. a non-human animal as hereinabove defined.

[0133] The polypeptides, compositions, nucleic acids and methods of the invention advantageously prevent and / or reduce ammonia release from animals and from animal excreta. The invention thereby contributes to reducing ammonia emissions from agriculture, and as such counteracts the negative consequences of such emissions for ecosystems and human and animal health. In addition, the invention also provides economic benefits, e.g., by preventing the loss of nitrogen from animal excreta through ammonia emissions, the resulting manure retains a high value as fertilizer for land.

[0134] An twelfth aspect relates a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for preventing or reducing the risk of ammonia poisoning of a non-human animal, for example by feeding or administrating the polypeptide or composition to the non-human animal. A preferred non- human animal is livestock, such as, but not limited to chickens, pigs, cows, sheep and goats,

[0135] An thirteenth aspect relates a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for preventing the infection of an urease-producing pathogen, such as, but not limited to, a Vibrio infection, preferably for the prevention of a Vibrio Parahaemolyticus, Vibrio Harveyi or Vibrio vulnificus infection, preferably by oral administration of the polypeptide. The polypeptide can be part of a composition as defined hereinabove. The polypeptide can be comprised in a feed as defined hereinabove. Hence the infection of an urease-producing pathogen can be prevented by feeding the animal a feed comprising the polypeptide with urease inhibiting activity. An fourteenth aspect relates a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for improving the shelf life of fish, for example by applying the polypeptide or composition on the skin of the fish. A preferred fish is selected from the group consisting of salmon, tuna, cod, tilapia, haddock, mackerel, sardine, halibut and catfish.

[0136] An fifteenth aspect relates a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein, preferably the polypeptide comprises an antigen-binding domain comprising or consisting of amino acid sequences of CDRs and / or ISVD as defined hereinabove, for use in the amelioration or treatment of a liver disease in a mammal, preferably a human. The polypeptide or composition is preferably provided by oral administration.

[0137] In a sixteenth aspect, there is therefore provided a method for producing a composition suitable for feeding or administering to a human, an (drinking) water supplement or a pharmaceutical composition comprising a polypeptide comprising an ISVD that specifically binds and inhibits a urease as described herein. The method comprises at least the step of admixing the polypeptide with further ingredients of the composition suitable for feeding or administering to a human, or pharmaceutical composition. The pharmaceutical composition may further comprise an excipient and / or pharmaceutical carrier,

[0138] The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

[0139] Description of the figures

[0140] Figure 1. Pepsin-resistance of VHH U616F as compared its unmodified parent VHH U61 F after 1 hr incubation at 37°C, at pepsin concentration as indicated. Buffer indicates control incubation pepsin buffer (pH 2.0) without pepsin.

[0141] Figure 2A. Set up of assay for urease inhibitory activity in extracts from plants or bacterial species as described in Example 4. Inhibitory activity of a VHH is compared to urease activity in the absence of the VHH (100%) and to background level of ammonia production in the absence of VHH and urease extract (0%).

[0142] Figure 2B. Set up of assay for urease inhibitory activity in extracts from plants or bacterial species as described in Example 4. Inhibitory activity of a VHH in faeces samples is compared to urease activity in the absence of the VHH (100%) and to background level of ammonia production in the absence of VHH and added urea (0%). Figure 3. Percentage urease inhibition by the various VHHs as indicated as determined in urease extracts from plants or bacteria as indicated, as described in Example 4.

[0143] Figure 4. Percentage urease inhibition by the various VHHs as indicated in faeces from a number of farm animals as indicated, as described in Example 4. The lowest row shows the average percentage inhibition over all the faeces samples in the above rows.

[0144] Examples 1 . Generation of modified urease-inhibiting VHHs

[0145] The original parent anti-urease VHHs, that are modified and / or used for comparison, are disclosed in co-pending application PCT / EP2024 / 053281 and are listed in Table 1 .1. Using standard molecular biological methods coding seguences for modified VHHs muteins as indicated in Table 1 .2 were and are prepared. Modified and parent VHHs were produced, purified and analysed essentially as described in in co-pending application PCT / EP2024 / 053281 . Production levels obtained in yeast for VHHs are indicated in the 2ndcolumn in Table 3.1 .

[0146] 2. Temperature stability of the modified urease-inhibiting VHHs

[0147] Temperature stability of original and modified urease-inhibiting was measured by incubation for 1 h at elevated temperatures and measuring antigen binding capacity in ELISA (data not shown). All VHHs resist one hour at 60°C incubation, some are slightly less active at 70°C while almost all VHHs are ~3 fold less active after 80°C incubation and some (U60F, U642F) even 10-20 fold lower. Temperature stability is thus not a major discriminating factor for VHHs.

[0148] 3. Protease-resistance of the modified urease-inhibiting VHHs

[0149] Protease-resistance of original and modified urease-inhibiting VHHs was determined by incubating the 1 pg of the VHHs for 1 hr at 37°C with either pepsin, trypsin or chymotrypsin in 10- fold dilution series of the proteases. Control incubations without protease in chymotrypsin buffer (pH 7.2) or in pepsin buffer (pH 2.0). Subseguently all samples were split in two and analysed on SDS-PAGE and by PAGE and by ELISA based on urease binding using a polyclonal anti-VHH antibody for VHH detection to calculate the residual percentage of VHH.

[0150] VHH U61 F and its 6 mutants, and U42F give low maximal absorbance values in these ELISAs and thus give unreliable percentages residual VHH in the ELISA assay (data not shown).

[0151] Table 3.1 summarizes that data from the VHH protease digestions. Figure 1 shows the strongly improved pepsin-resistance of VHH U616F as compared its unmodified parent VHH U61 F. The main conclusions from the data are follows.

[0152] Parent VHH U61 F is mostly degraded by 0.01 mg / ml pepsin, but several of its mutants show higher pepsin resistance, especially U612F (band at 0.1 mg / ml visible) and U616F (band at 1 mg / ml visible). Trypsin and chymotrypsin resistance is unaltered. A stable 17 kDa trypsin product is present in parent VHH U61 F and in all of its 6 mutants.

[0153] For VHH U49F a band is still visible at 0.01 mg / ml pepsin. U49F also shows good chymotrypsin resistance but lower trypsin resistance.

[0154] Parent VHH U64F shows only 4% residual at 0.01 mg / ml pepsin but a stable trypsin product at 16 kDa that then decreases to 14 kDa at higher trypsin concentration. U64F shows reasonable (>20%) trypsin and chymotrypsin resistance. Several U64F mutants show increased pepsin resistance. Most notably U642F and U643F, which show VHH bands at 1 mg / ml pepsin. In case of U642F trypsin resistance improved (band stays at 16 kDa) and chymotrypsin remained good while in case of U643F chymotrypsin resistance went down. Protease resistance of mutant U644F is higher than that of its parent U64F. pH 2 represents the lower range of acidic conditions in the stomach of animals. All tested VHH’s are resistant against a pH 2 environment as all of them survived the control part of the pepsin experiment. 4. Urease inhibitory activity of the modified VHHs

[0155] Urease inhibitory activity of the modified VHHs against a variety of plant and bacterial ureases was compared to those of several unmodified VHHs. In addition, in situ urease inhibitory activity of the modified and unmodified VHHs was determined in faeces of different animals.

[0156] For determining urease inhibitory activity against bacterial ureases, bacterial strains of the species Proteus mirabilis, Escherichia coli, Klebsiella aerogenes, Providencia rettgeri, and Morganella morganii were selected partly based on phylogeny (see Konieczna et al., 2012, Curr Protein Pept Sci 13, 789-806). Bacterial ureases were obtained by culturing cells, washing, sonication in 20 mM Hepes pH 6.5 and centrifugation. Extracts were tested for urease activity and diluted to a concentration suitable for the inhibition assay, indicated in larger type while red text indicates commercial purified ureases.

[0157] Soy (Glycine max) urease was obtained from soy beans by grinding in a mortar using 20 mM Hepes pH 6.5 and centrifugation, the extracts were tested for urease activity and diluted to a concentration suitable for the inhibition assay. Jack Bean (Canavalia ensiformis) urease was obtained commercially (Sigma Aldrich, U4002-100KU) and diluted to a concentration suitable for the inhibition assay.

[0158] Faeces was obtained from animals as indicated in Figure 4 and diluted in 20 mM Hepes pH 6.5 to a concentration suitable for the inhibition assay.

[0159] Urease inhibitory activity of the VHHs in faeces and in the plant and bacterial extracts was determined using the Phenol red assay, essentially as described in co-pending application PCT / EP2024 / 053281 . The set up for determining inhibitory activity of the VHHs in bacterial or plant extracts is shown in Figure 2A, and for determining inhibitory activity of the VHHs in faeces is shown in Figure 2B. For the inhibition of the P. rettgeri and P. mirabilis urease inhibition the x-axis intercept was set at -40% to compensate for negative inhibition observed. The three negative control nonurease VHHs M98F, M170F, M181 F at the right also show such negative inhibition of about 40%.

[0160] Figure 3 shows the results of the urease inhibitory activity of the various VHHs against a variety of plant and bacterial ureases. U49F and U61 F and derivatives show that broadest inhibitory action: U49F shows inhibitory activity against all seven ureases tested and VHHs in the U61 F-family inhibit at least five out of the seven ureases tested. VHHs in the U64F-family show inhibition of the plant ureases and of three bacterial urease but do not inhibit two further bacterial ureases. Interestingly, the bacterial inhibition spectra of the U61 F- and U64F-families seem complementary in that bacterial urease that are not inhibited by the U61 F-family are inhibited by the U64F-family and vice versa.

[0161] Figure 4 shows the results for the in situ urease inhibitory activity of the various VHHs in faeces of various farm animals. The bottom row in Figure 4 show the average inhibition over the various faeces samples tested, which averages are also presented in Table 4.1 .

[0162] Up to 100% inhibition in some of the faces samples is seen with a number of VHHs. The highest average percentage inhibition in faeces, combined with a good protease resistance, is seen for U49F and for U61 F and its derivatives. Also U64F and its derivatives combine good protease resistance with reasonable percentages average inhibition in faeces. Table 3.1 . VHH production levels in yeasts and a summary of the protease digestion data for the

[0163] VHHs as indicated. aFor U61 F, its 6 mutants and U42F, that were unreliable for ELISA, SDS-PAGE data are given for pepsin and trypsin. Table 3.1 (Continued). aFor U61 F, its 6 mutants and U42F, that were unreliable for ELISA, SDS-PAGE data are given for pepsin and trypsin. Table 4.1 . Average percentage of urease inhibition by VHH as indicated over faeces samples from different animals as indicated in Figure 4.

[0164] 4. Pepsin, Chymotrypsin, and Trypsin Treatments followed by Phenol Red Assay

[0165] Material and methods Tested VHHs:

[0166] • U492 (U49.2)

[0167] • U618 (U61.8)

[0168] • U648 (U64.8) Table 5.1. Amino acid sequence of tested VHHs

[0169] Table 5.2. Amino acid sequence of parent and tested VHHs, IMGT aligned*

[0170] * The annotation in the amino acid sequence is to indicate the alignment with IMGT, i.e. the sequence is a continuous amino acid sequence.

[0171] Table 5.3. Mutations introduced in parent VHHs and isoelectric point

[0172] Pepsin Treatment

[0173] • 2mg / ml pepsin solution was prepared in 20mM HCI

[0174] • Dilution series: i. 2mg / ml in 20mM HCI ii. 0.2mg / ml in 20mM HCI

[0175] Hi. 0.02mg / ml in 20mM HCI iv. 0.002mg / ml in 20mM HCI v. Blank: Omg / ml in 20mM HCI

[0176] • A premix of VHH in water was prepared: i. 72pl VHH at 1 mg / ml (6*12pl) ii. 78pl H2O (6*13pl)

[0177] Hi. Dispense 25pl per well.

[0178] • 25p I was added from the dilution series enzyme to the prepared VHHs

[0179] • Incubated 1 hr at 37°C

[0180] Neutralization

[0181] • 25pl treated VHH was added to 25ul 10mM NaOH : 10 minutes 70°C incubation.

[0182] • Incubated 15 min on ice

[0183] • Added 50pl 40mM HEPES.

[0184] Continued with Phenol Red Assay to determine the percentage of inhibition Chymotrypsin treatment

[0185] • Chymotrypsin dilution series prepared in 2mM Tris-HCI pH8 + 40mM CaCI2 i. 2mg / ml ii. 0.2mg / ml

[0186] Hi. 0.02mg / ml iv. Blank: Omg / ml

[0187] • A premix of VHH in water was prepared: i. 95.04pl VHH at 1 mg / ml (6*15.84pl) ii. 3.96pl H2O (6*0.66pl)

[0188] Hi. Dispensed 16.5pl per well tested

[0189] • 16.5pl from the dilution series was added to each VHH mix

[0190] • Incubated 1 hr at 37°C

[0191] Neutralization

[0192] • 12.5pl 20mM HCI was added per sample

[0193] • Incubated 1 hr on ice

[0194] • 25pl 10mM NaOH was added

[0195] • 50pl 40mM HEPES was added

[0196] Trypsin treatment

[0197] • Dilution series of trypsin was prepared in 2mM Tris-HCI pH8 + 40mM CaCI2 i. 2mg / ml ii. 0.2mg / ml

[0198] Hi. 0.02mg / ml iv. Blank: Omg / ml

[0199] • A premix of VHH in water was prepared: i. 95.04pl VHH at 1 mg / ml (6*15.84pl) ii. 3.96pl H2O (6*0.66pl)

[0200] Hi. Dispense 4x 16.5pl per well

[0201] • 25ul from the dilution series was added to each VHH mix

[0202] • Incubated 1 hr at 37°C

[0203] Neutralization

[0204] • 12.5pl 20mM HCI was added per sample

[0205] • Incubated 1 hr on ice

[0206] • 25pl 10mM NaOH was added

[0207] • 50pl 40mM HEPES was added.

[0208] Continued with Phenol Red Assay to determine the percentage of inhibition. Phenol red assay.

[0209] • Jack Bean Urease solution (JBUS) was prepared (1 pg / ml in 20mM HEPES)

[0210] • 25pl treated VHHs was added into designated wells (triplicates)

[0211] • 24pl 20mM HEPES + 1 pl untreated VHH in was added to control wells

[0212] • 50pl JBUS solution was added to designated wells

[0213] • Control sample: 75pl 20mM HEPES was used

[0214] • Incubated 1 hr at 37°C

[0215] • 300pl Phenol Red, 468.75pl 8M Urea, 6731 ,25pl 20mM HEPES was added to the samples

[0216] • Covered and started continuous measurement in Spectrostarto follow OD development.

[0217] Results:

[0218] Table 6. 1. Inhibition at 180 minutes for urease inhibitor (VHH) U492K (percentage inhibition)

[0219] Table 6.2. Inhibition at 180 minutes for urease inhibitor (VHH) U618K (percentage inhibition)

[0220] Table 6.3. Inhibition at 180 minutes for urease inhibitor (VHH) 648K (percentage inhibition)

[0221] Conclusion:

[0222] VHH 492 and 618 are surprisingly resistant against chymotrypsin and trypsin treatment. VHH 648 is slightly less resistant regarding these treatments. All three VHH are increasingly sensitive to increasing concentrations of pepsin.

Claims

Claims1 . A polypeptide comprising an immunoglobulin single variable domain (ISVD) that specifically binds and inhibits a urease, wherein: a) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 7; b) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises at least one modification selected from the group consisting of Q1 E, Q5V, A15P, R90Q, A96P, D97E, S100A and A54C - I78C, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 10; or c) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the ISVD comprises at least one modification selected from the group consisting of R28F, A54C - I78C, Q1 E, Q5V and A15P, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 3.

2. A polypeptide according to claim 1 , wherein: a) the ISVD comprising CDRs as set forth in SEQ ID NO: 7, comprises a modification or a combination of modifications selected from the group consisting of: i) Q1 E and Q5V; ii) K84T; iii) R108Q; iv) A54C - I78C; v) Q1 E, Q5V and K84T; vi) Q1 E, Q5V and R108Q; vii) Q1 E, Q5V and A54C - I78C; viii) Q1 E, Q5V, K84T and R108Q; ix) Q1 E, Q5V, K84T and A54C - I78C; x) Q1 E, Q5V, R108Q and A54C - I78C; xi) K84T, R108Q and A54C - I78C; and xii) Q1 E, Q5V, K84T, R108Q and A54C - I78C; b) the ISVD comprising CDRs as set forth in SEQ ID NO: 10, comprises a modification or a combination of modifications selected from the group consisting of: i) A54C - I78C; ii) Q1 E, Q5V and A15P; iii) Q1 E and Q5V; iv) R90Q, A96P, D97E and S100A; v) Q5V, R90Q, A96P, D97E and S100A; vi) Q1 E, Q5V, R90Q, A96P, D97E and S100A; vii) Q1 E, Q5V, R90Q, A96P and D97E; viii) Q1 E, Q5V, R90Q, A96P and S100A; ix) Q1 E, Q5V, R90Q, D97E and S100A; x) Q1 E, Q5V, A96P, D97E and S100A; xi) Q1 E, Q5V, R90Q and A96P; xii) Q1 E, Q5V, R90Q and D97E; xiii) Q1 E, Q5V, R90Q and S100A; xiv) Q1 E, Q5V, A96Pand D97E; xv) Q1 E, Q5V, A96P and S100A; xvi) Q1 E, Q5V, D97E and S100A; xvii) Q1 E, Q5V and R90Q; xviii) Q1 E, Q5V and A96P; xix) Q1 E, Q5V and D97E; xx) Q1 E, Q5V and S100A; and, xxi) Q1 E, Q5V, R90Q, A96P, D97E, S100A and A54C - I78C; or c) the ISVD comprising CDRs as set forth in SEQ ID NO: 3, comprises a modification or a combination of modifications selected from the group consisting of: i) R28F; ii) A54C - I78C; iii) R28F and A54C - I78C; iv) Q1 E, Q5V and A15P; v) Q1 E and Q5V; vi) Q1 E, Q5V and R28F; and, vii) Q1 E, Q5V, R28F and A54C - I78C.

3. A polypeptide according to claim 1 or 2, wherein a) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the ISVD comprises the mutation Q1 E, Q5V, K84T and R108Q, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 7; b) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10, wherein the ISVD comprises the mutations Q1 E, Q5V, R90Q, A96P and S100A, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 10; or c) the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:

3. wherein the ISVD comprises the mutations Q1 E, Q5V and R28F, and wherein the ISVD comprises CDRs that differ by no more than 2 amino acids from the CDRs as set forth in SEQ ID NO: 3.

4. A polypeptide according to any one of the preceding claims, wherein: a) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 7, except for the at least one modification or combination of modifications defined in a) of any one of claims 1 - 3; b) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 10, except for the at least one modification or combination of modifications defined in b) of any one of claims 1 - 3; or, c) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 3, except for the at least one modification or combination of modifications defined in c) of claim 1 or 2.

5. A polypeptide according to any one of the preceding claims, wherein: a) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 21 ;b) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 22; or, c) the ISVD comprises an amino acid sequence as set forth in SEQ ID NO: 23.

6. A polypeptide according to any one of the preceding claims, wherein the CDRs are Kabat CDRs, Chothia CDRs or IMGT CDRs.

7. A polypeptide comprising more than one ISVD as defined in any one of the preceding claims, wherein, optionally the ISVDs are linked through a spacer amino acid sequence, wherein preferably the polypeptide comprising at least two different ISVDs.

8. A polypeptide according to claim 6, wherein the polypeptide comprises: a) a first ISVD that is an ISVD as defined in claims 1 - 6, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, and a second ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10; b) a first ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, and a second ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3; c) a first ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 10, and a second ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3; or, d) a first ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 7, a second ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or2 amino acids from the CDRs as set forth in SEQ ID NO: 10, and a third ISVD that is an ISVD as defined in claims 1 - 4, that has CDRs that differ by no more than 1 or 2 amino acids from the CDRs as set forth in SEQ ID NO: 3.

9. A composition comprising a polypeptide as defined in any one of the preceding claims, wherein preferably, the composition is suitable for feeding or administering to a non-human animal.

10. A composition according to claim 9, wherein the composition comprises more than one different polypeptides as defined in any one of the preceding claims.

11. A composition according to claim 9 or 10, wherein the composition comprises a polypeptide as defined in any one of the preceding claims in combination with a polypeptide comprising an ISVD that comprises CDRs that differ by no more than 2 amino acids from the CDRs asset forth in a sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 -23.

12. A composition according to claim 11 , wherein the ISVD comprises an amino acid sequence with at least 91 %, or with increasing preference, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence as set forth in the sequence selected from the group consisting of SEQ ID NO.’s: 1 - 17 and 21 - 23.

13. A nucleic acid encoding a polypeptide as defined in any one of claims 1 - 8.

14. A method for inhibiting urease activity in the gastro-intestinal tract of an animal, wherein the method comprises feeding or administering to the animal a polypeptide as defined in any one of claims 1 - 8 or a composition as defined in any one of claims 9 - 12.

15. A method for reducing at least one of the amount of ammonia released from an animal and the amount of ammonia released from the animal excreta, wherein the method comprises feeding or administering to the animal a polypeptide as defined in any one of claims 1 - 8 or a composition as defined in any one of claims 9 - 12.

16. Use of a polypeptide as defined in any one of claims 1 - 8 or a composition as defined in any one of claims 9 - 12, for at least one of: a) reducing the amount of ammonia released from an animal, wherein, preferably the amount of ammonia released from an animal is reduced by inhibiting urease activity in the gastro-intestinal tract of the animal; b) reducing the amount of ammonia released from the excreta of animal, wherein, preferably the amount of ammonia released from the animal’s excreta is reduced by inhibiting urease activity in the animal’s excreta, more preferably in the feces of the animal; c) reducing the amount of ammonia released from animal feeding operations; preferably for reducing the amount of ammonia released from feeding operations into the atmosphere; d) preventing the loss of nitrogen-value in manure; e) reducing ammonia volatilization from the use of urea as nitrogen fertilizer; and, f) reducing the amount of ammonia released from a surface comprising urease from microbial or vegetal sources, for example by applying the composition or polypeptide onto the surface.

17. Use of a polypeptide as defined in any one of claims 1 - 8 or a composition as defined in any one of claims 9 - 12, for reducing the amount of ammonia released from an animal’s excreta by applying a composition comprising the polypeptide onto the animal’s excreta.

18. A polypeptide as defined in any one of claims 1 - 8 or a composition as defined in any one of claims 9 - 12, for use in the prevention or treatment of hyperammonemia.

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