WO2024165710A1

WO2024165710A1 - Immunoglobulin single variable domains that inhibit urease and use thereof

Info

Publication number: WO2024165710A1
Application number: PCT/EP2024/053281
Authority: WO
Inventors: Gerrit Dirk Keizer; Michaël Marie HARMSEN
Original assignee: Seni-Preps B.V.
Priority date: 2023-02-09
Filing date: 2024-02-09
Publication date: 2024-08-15

Abstract

The present invention relates to polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity, preferably, the antigen-binding domain inhibits the activity of plant and/or bacterial 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 ecosystems.

Description

Immunoglobulin single variable domains that inhibit urease and use thereof

Field of the invention

The present invention relates to the field of immunology, enzymology, agriculture and animal husbandry. In particular, the invention relates to polypeptides comprising immunoglobulin single variable domains (ISVDs) that inhibit the enzymatic activity of ureases. The invention further relates the use of such polypeptides for reducing ammonia release from animals and from animal excreta in e.g. the stables and slurry pit.

Background of the invention

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.

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.

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).

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.

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.

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.

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

Helicobacter pylori for use in the treatment of H. pylori infection.

It is an object of the present invention to provide for means and methods for reducing ammonia release from animals and from animal excreta.

Summary of the invention

In a first aspect, there is provided a composition suitable for feeding or administering to a non-human animal comprising a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity.

In one embodiment, there is provided a composition suitable for feeding or administering to a non-human animal comprising a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity. In one embodiment, the antigen-binding domain is an immunoglobulin single variable domain (ISVD). In one embodiment, the composition is for reducing at least one of the amount of ammonia released from the animal and the amount of ammonia released from the animal’s excreta. In one embodiment, the composition is an animal feed, an animal feed premix, an animal feed supplement or an animal drink composition.

In one embodiment, the composition is a composition wherein the antigen-binding domain inhibits at least one of a plant urease and a bacterial urease, preferably the antigen-binding domain inhibits a plant urease and a bacterial urease. In one embodiment, the plant urease is a Jack Bean urease and the bacterial urease is a urease from Klebsiella aerogenes or Helicobacter pylori. In one embodiment, the composition is a composition wherein the antigen-binding domain crossblocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 1 - 11 to at least one of a plant and a bacterial urease and/or wherein the antigen-binding domain is cross-blocked from binding at least one of a plant and a bacterial urease by at least one of the VHHs.

In one embodiment, the composition is a composition wherein the antigen-binding domain binds to or near the FLAP region of at least one of the plant urease and the bacterial urease.

In one embodiment, there is provided a composition comprising a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a microbial urease. In one embodiment, the plant urease is a Jack Bean urease and the microbial urease is a bacterial urease, wherein preferably, the bacterial urease is a urease from Klebsiella aerogenes. In one embodiment, at least one of: i) the first polypeptide inhibits at least 20% activity of the plant urease; and, ii) the second polypeptide inhibits at least 20% activity of the microbial urease, preferably when assayed at a twofold molar excess of the antigen-binding domain.

In one embodiment, the composition is a composition wherein at least one of the first and second polypeptides do not specifically bind to a urease from Helicobacter pylori.

In one embodiment, the composition is a composition wherein: i) the first antigen-binding domain cross-blocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 3, 7, 10 and 9, to the plant urease and/or wherein the first antigen-binding domain is cross-blocked from binding the plant urease by at least one of the VHHs; and ii) the second antigen-binding domain cross-blocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 61 - 65, to the microbial urease and/or wherein the second antigen-binding domain is cross-blocked from binding the microbial urease by at least one of the VHHs.

In one embodiment, the composition is a composition wherein the first antigen-binding domain is a first ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 15, 16, or 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 18, 19, or 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 28, 29, or 30; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 31 , 32, or 33; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 35; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 36; and wherein the second antigen-binding domain is a second ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 66 or 67, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 66 or 67; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 68 or 69 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 68 or 69; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 71 , or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 71 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 72 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 72; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 73 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 73; e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 74, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 74; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 75 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 75; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 76 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 76; f) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 77, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 77; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 78 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 78; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 79 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 79.

In one embodiment, the composition is a composition wherein the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NOs: 20; and, b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NOs: 25, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 25; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 28, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 28; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 31 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 31 ; c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 35; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 36; and wherein the second ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 66, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.’s: 66; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 68 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.’s: 68; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70.

In one embodiment, the composition is a composition wherein the first ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: a) an amino acid sequence as set forth in SEQ ID NO.’s: 1 , 2, or 3; b) an amino acid sequence as set forth in SEQ ID NO.’s: 7, 8, or 9; or c) an amino acid sequence as set forth in SEQ ID NO: 10; and wherein the second ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: c) an amino acid sequence as set forth in SEQ ID NO.’s: 61 or 62; d) an amino acid sequence as set forth in SEQ ID NO: 63; e) an amino acid sequence as set forth in SEQ ID NO: 64; or, f) an amino acid sequence as set forth in SEQ ID NO: 65.

In one embodiment, the composition is a composition comprising a bispecific polypeptide wherein the first and second polypeptides are fused in a single polypeptide chain, wherein, optionally the first and second polypeptides are linked through a spacer amino acid sequence.

In a second aspect, there is provided a polypeptide as defined in the first aspect herein.

In one embodiment, the polypeptide comprises an anti-urease antigen-binding domain that inhibits urease activity, wherein the antigen-binding domain is an ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 15, 16, or 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 18, 19, or 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 21 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 21 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 22, 23 or 88, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 22, 23 or 88; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 24, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 24; c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequences of SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 28, 29, or 30; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 31 , 32, or 33; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 35; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 36; and, e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 37; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 38; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 39, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 39.

In one embodiment, the ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 21 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 22, 23 or 88; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 24; c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36; and, e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 37; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 38; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 39. In one embodiment, the ISVD comprises an amino acid sequence with at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 or 100% sequence identity with: a) the amino acid sequence as set forth in SEQ ID NO.’s: 1 , 2, or 3; b) the amino acid sequence as set forth in SEQ ID NO.’s: 4, 5, or 6; c) the amino acid sequence as set forth in SEQ ID NO.’s: 7, 8, or 9; d) the amino acid sequence as set forth in SEQ ID NO.: 10; and, e) the amino acid sequence as set forth in SEQ ID NO.: 11 .

In one embodiment, the polypeptide comprises more than one anti-urease antigen-binding domain, optionally linked through a spacer amino acid sequence. In one embodiment, the polypeptide is a polypeptide that is protease resistant. In one embodiment, 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, 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 the examples.

In a third aspect, there is provided a nucleic acid encoding the polypeptide as defined in the first or second aspect.

In a fourth aspect, there is provided a method for inhibiting urease activity in the intestines or gastrointestinal tract of an animal, wherein the method comprises feeding or administering to the animal a composition comprising a polypeptide as defined in the second aspect.

In a fifth 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 as defined in the second aspect.

In a sixth aspect, there is provided a use of a composition as defined in the first aspect, or a polypeptide 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 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; and, e) reducing ammonia volatilization from the use of urea as nitrogen fertilizer.

In a seventh aspect, there is provided a use of a polypeptide as defined in the first or 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.

In an eighth aspect, there is provided a polypeptide as defined in the second aspect, for use in the prevention and/or treatment of a Helicobacter pylori infection.

Description of the invention

Definitions

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.

“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.

“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.

“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.

“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.

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

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.

“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.

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.

Alternative conservative amino acid residue substitution classes.

Alternative physical and functional classifications of amino acid residues.

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.

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.

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)).

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.

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.

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.

Table A. CDR defintions¹

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).

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).

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.

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.

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.

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.

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.

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 10'⁶ to 10'¹² M or less, and preferably 1 O’⁸ to 1 O’¹² M or less, and/or with a binding affinity of at least 10^-6 M or 10^-7 M, preferably at least 10^-8 M, more preferably at least 10^-9 M, such as at least 10’¹°, 10^-11, 10^-12 M or more. Any KD value greater than 10^-4 M (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’ ⁴ M, 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.

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 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 (k_on) 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 10⁶ M^-1 S^-1 by 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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

Detailed description of the invention

The present invention relates to novel 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 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 polypeptides with urease inhibiting activity can be applied (ex vivo) to the animal’s excreta to prevent and/or reduce ammonia release therefrom.

In a first aspect therefore, there is provided a composition suitable for feeding or administering to a non-human animal comprising a polypeptide comprising an anti-urease antigenbinding domain that inhibits urease activity.

In one embodiment, there is provided a composition comprising at least one of a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a microbial urease.

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 comprising a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity 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.

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, and equine species. By way of example the non-human animals can include cattle, poultry, chickens, turkeys, ducks, quail, geese, pigs, goats and sheep.

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.

A second aspect pertains to a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity. The polypeptide can be comprised in an animal feed, an animal feed premix or an animal feed supplement as described herein. 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 as described in the Examples herein.

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.

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 a Jack bean (Canavalia ensiformis) urease.

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.

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 and Lactobacillus. In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a Klebsiella aerogenes urease, preferably the antigen-binding domain inhibits at least a Klebsiella aerogenes urease.

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.

In one embodiment, the first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease does not specifically bind to a urease from Helicobacter pylori. In one embodiment, the second polypeptide comprising a second anti-urease antigenbinding domain that inhibits activity of a microbial urease does not specifically bind to a urease from Helicobacter pylori. In one embodiment, at least one of the first and second polypeptides do not specifically bind to a urease from Helicobacter pylori.

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%, 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, the first polypeptide comprises a first anti-urease antigen-binding domain that inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 90%, 99% or 100% activity of a plant urease as defined above, when assayed at a twofold molar excess of the antigenbinding domain. In one embodiment, the second polypeptide comprises a second anti-urease antigen-binding domain that inhibits at least 10%, or with increasing preference 20%, 50%, 75%, 90%, 99% or 100% activity of a microbial 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 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 (first) 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 (second) 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.

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.

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 (first) 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 (second) polypeptide as described herein comprises an antigenbinding 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.

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.

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 the Examples herein.

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain with the ability to cross-block the binding of at least one VHH disclosed herein to at least one of a plant and a bacterial urease as defined above and/or to be cross-blocked from binding a plant and a bacterial urease as defined above by at least one VHH disclosed herein. Thus, in one embodiment the polypeptide comprise an antigen-binding domain that cross-blocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 1 - 11 to at least one of Jack Bean and Klebsiella aerogenes ureases, and/or that is cross-blocked from binding to at least one of Jack Bean and Klebsiella aerogenes ureases by of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 1 - 11. In one embodiment, a first polypeptide as described herein comprises a first antigen-binding domain which has the ability to cross-block the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 3, 7, 10 and 9, preferably at least one of SEQ ID NO.’s: 3, 7 and 10, more preferably at least one of SEQ ID NO.’s: 3 and 7, and most preferably SEQ ID NO: 3, to a plant urease as defined above and/or the first antigen-binding domain can be cross-blocked from binding the plant urease by at least one of these VHHs. In one embodiment, a second polypeptide as described herein comprises a second antigen-binding domain which has the ability to cross-block the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 61 - 65, preferably at least one of SEQ ID NO.’s: 61 , 62 and 63, more preferably at least one of SEQ ID NO.’s: 61 and 62, and most preferably SEQ ID NO: 61 , to a microbial or bacterial urease as defined above and/or the second antigen-binding domain can be cross-blocked from binding the plant urease by at least one of the VHHs.

Assays for determining competition between different antigen-binding domains for binding to the same antigen or epitope are well known in the art and can be performed as described in the Examples herein.

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that binds to or near the FLAP region of at least one of a plant and a bacterial urease as defined above. Preferably, the antigen-binding domain binds at least to or near the FLAP region of Jack Bean urease. The ability of an antigen-binding domain to bind to the FLAP region of Jack Bean urease may be assayed by assessing the ability of the antigen-binding domain to bind to a synthetic peptide having the amino acid sequence of SEQ ID NO: 47, as described in the Examples herein.

An example of an antigen-binding domain that binds to the FLAP region of Jack Bean urease is an antigen-binding domain having a combination of complementarity-determining regions (CDRs) of CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 21 , a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 23 and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 24. Preferably, an antigen-binding domain that binds to the FLAP region of Jack Bean urease is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least

82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98%, at least 99 or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5. Thus, a VHH comprising an amino acid sequence as set forth in SEQ ID NO: 5 can serve as a reference antigen-binding domain that binds to the FLAP region of Jack Bean urease.

The ability of an antigen-binding domain to bind to or near the FLAP region of at least one of a plant and a bacterial urease as defined above, is herein defined as the ability of the antigenbinding domain to cross-block the binding of a reference antigen-binding domain to at least one of a plant and a bacterial urease and/or to be cross-blocked from binding a plant and a bacterial urease by the reference antigen-binding domain, wherein the reference antigen-binding domain is an antigen-binding domain that is known to bind to the FLAP region of at least one of a plant and a bacterial urease, such as e.g. the VHH comprising an amino acid sequence as set forth in SEQ ID NO: 5. Thus, in one embodiment, a polypeptide as described herein comprises an antigen-binding domain that binds to or near the FLAP region of Jack Bean urease, wherein the antigen-binding domain cross-blocks the binding of a VHH comprising an amino acid sequence as set forth in SEQ ID NO: 5 to Jack Bean urease and/or wherein the antigen-binding domain is cross-blocked from binding Jack Bean urease by the VHH. In a preferred embodiment, the antigen-binding domain cross-blocks the binding of the VHH comprising the amino acid sequence as set forth in SEQ ID NO: 5 to a synthetic peptide having the amino acid sequence of SEQ ID NO: 47 and/or the antigenbinding domain is cross-blocked from binding a synthetic peptide having the amino acid sequence of SEQ ID NO: 47 by the VHH.

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.

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease as defined above, wherein the antigen-binding domain is an ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequences of SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17 or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequences of SEQ ID NO.’s: 15, 16, or 17; and, a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 18, 19, or 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 21 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 21 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 22, 23 or 88, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 22, 23 or 88; and, a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 24, or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 24; c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 28, 29, or 30; and, a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 31 , 32, or 33; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 35; and, a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 36; and, e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 37; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 38; and, a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 39, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 39.

In a preferred embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease as defined above, wherein the antigen-binding domain is an ISVD comprising a CDR combination of CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33;

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease as defined above, wherein the antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least

78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least

85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least

92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

99% or 100% sequence identity with: a) the amino acid sequence as set forth in SEQ ID NO.’s: 1 , 2, or 3; b) the amino acid sequence as set forth in SEQ ID NO.’s: 4, 5, or 6; c) the amino acid sequence as set forth in SEQ ID NO.’s: 7, 8, or 9; d) the amino acid sequence as set forth in SEQ ID NO: 10; and, e) the amino acid sequence as set forth in SEQ ID NO: 11. In a preferred embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease as defined above, wherein the antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth, with decreasing preference, in SEQ ID NO.’s,: 3, 7, 10, or 9. In a more preferred embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a plant urease as defined above, wherein the antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least

83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO.’s: 3 or 7, of which 3 is most preferred.

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a bacterial urease as defined above, wherein the antigen-binding domain is an ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 66 or 67, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 66 or 67; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 68 or 69 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 68 or 69; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 71 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 71 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 72 or an amino acid sequence that has 2 or 1 amino acid difference^) with the amino acid sequence of SEQ ID NO: 72; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 73 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 73; c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 74, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 74; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 75 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 75; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 76 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 76; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 77, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 77; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 78 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 78; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 79 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 79.

In a preferred embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a bacterial urease as defined above, wherein the antigen-binding domain is an ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 66, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 66; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 68 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO: 68; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70;

In one embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a bacterial urease as defined above, wherein the antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least

99% or 100% sequence identity with, with decreasing preference: a) an amino acid sequence as set forth in SEQ ID NO.’s: 61 or 62; b) an amino acid sequence as set forth in SEQ ID NO: 63; c) an amino acid sequence as set forth in SEQ ID NO: 64; and, d) an amino acid sequence as set forth in SEQ ID NO: 65. In a preferred embodiment, a polypeptide as described herein comprises an antigen-binding domain that inhibits at least a bacterial urease as defined above, wherein the antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least

76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least

97%, at least 98%, at least 99% or 100% sequence identity with: a) an amino acid sequence as set forth in SEQ ID NO: 61 .

In one embodiment, there is provided a composition comprising a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a bacterial urease, wherein the first antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with: a) the amino acid sequence as set forth in SEQ ID NO.’s: 1 , 2, or 3; b) the amino acid sequence as set forth in SEQ ID NO.’s: 4, 5, or 6; c) the amino acid sequence as set forth in SEQ ID NO.’s: 7, 8, or 9; d) the amino acid sequence as set forth in SEQ ID NO: 10; and, e) the amino acid sequence as set forth in SEQ ID NO: 11 , and wherein the second antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or

100% sequence identity with: f) an amino acid sequence as set forth in SEQ ID NO.’s: 61 or 62; g) an amino acid sequence as set forth in SEQ ID NO: 63; h) an amino acid sequence as set forth in SEQ ID NO: 64; and, i) an amino acid sequence as set forth in SEQ ID NO: 65.

In a preferred embodiment, there is provided a composition comprising a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a bacterial urease, wherein the first antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO.’s: 3, 7, 10 or 9, of which preferably SEQ ID NO.’s: 3 or 7 of which SEQ ID NO: 3 is most preferred, and wherein the second antigen-binding domain is an ISVD comprising an amino acid sequence with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with: with an amino acid sequence as set forth in SEQ ID NO.’s: 61 or 62, of which SEQ ID NO: 61 is preferred.

Throughout this disclosure it is preferred that the same percent values are selected for the sequence identity of combinations of more than one antigen-binding domains, such as e.g. a combination of a first and a second anti-urease antigen-binding domains.

In one embodiment, there is provided a composition comprising a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a bacterial urease, wherein the first and second antigen-binding domain are ISVDs comprising amino acid sequences with at least 70%, or with increasing preference, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least

80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with: SEQ ID NO.’s: 3 and 61 , respectively; SEQ ID NO.’s: 3 and 62, respectively; 3 and 63, SEQ ID NO.’s: 3 and 64, respectively; SEQ ID NO.’s: 3 and 65, respectively; SEQ ID NO.’s: 7 and 61 , respectively; SEQ ID NO.’s: 7 and 62, respectively; SEQ ID NO.’s: 7 and 63, respectively; SEQ ID NO.’s: 7 and 64, respectively; SEQ ID NO.’s: 7 and 65, respectively; SEQ ID NO.’s: 9 and 61 , respectively; SEQ ID NO.’s: 9 and 62, respectively; SEQ ID NO.’s: 9 and 63, respectively; SEQ ID NO.’s: 9 and 64, respectively; SEQ ID NO.’s: 9 and 65, respectively; SEQ ID NO.’s: 10 and 61 , respectively; SEQ ID NO.’s: 10 and 62, respectively; SEQ ID NO.’s: 10 and 63, respectively; SEQ ID NO.’s: 10 and 64, or SEQ ID NO.’s: 10 and 65 respectively.

In one embodiment, a polypeptide as described herein comprises more than one anti-urease antigen-binding domain as described herein, optionally linked through a spacer amino acid sequence. Thus, antigen-binding 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. In a preferred embodiment, there is provided a bispecific polypeptide comprising a first polypeptide as described herein, comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide as described herein, comprising a second anti-urease antigen-binding domain that inhibits activity of a bacterial urease, wherein the first and second polypeptides 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.

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.

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 the sequence of (Gly-Gly-Gly-Gly-Ser)n. 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: 48) and EGKSSGSGSESKST (SEQ ID NO: 50), that have been applied for the construction of a bioactive scFv’s.

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. The anti-urease antigen-binding domain in a polypeptide as described herein therefore preferably is an ISVD, which are known to be more resistant to proteolytic degradation. Protease resistant ISVDs can be obtained by screening panels of ISVDs for susceptibility to proteolytic degradation (see e.g. Harmsen et al., 2006 and Maffey et al., 2016), and/or proteolytic stability of ISVDs can be improved by random mutagenesis approaches or rational protein engineering (see e.g. Harmsen et al., 2006; Hussack et al., 2011 ; Hussack et al., 2014; and Rutten et al., 2012). A preferred protease resistant anti-urease ISVD has an He residue at IMGT position 28, instead of the Arg residue that often occurs at this position in ISVDs.

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).

In a third aspect, there is provided a nucleic acid encoding a polypeptide comprising an antiurease antigen-binding domain that inhibits urease activity 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. Examples of nucleotide sequences encoding a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity as described herein that are codon-optimized for expression in Saccharomyces cerevisiae are given in SEQ ID NO.’s: 50 - 60.

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 a nucleic acid molecule 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).

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 anti-urease antigen-binding domain that inhibits urease activity a described herein. The method comprises at least the step of ad mixing the polypeptide with further ingredients of the composition suitable for feeding or administering to a non-human animal, or pharmaceutical composition.

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 anti-urease antigen-binding domain that inhibits urease activity 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.

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 anti-urease antigen-binding domain that inhibits urease activity 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.

A nineth aspect relates to use of a composition comprising a polypeptide comprising an antiurease antigen-binding domain that inhibits urease activity 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.

In a tenth aspect, there is provided use of a polypeptide comprising an anti-urease antigenbinding domain that inhibits urease activity 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 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.

An eleventh aspect relates a polypeptide comprising an anti-urease antigen-binding domain that inhibits urease activity 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 use in the prevention and/or treatment of a Helicobacter pylori infection. The polypeptide can be used for the prevention and/or treatment of a Helicobacter pylori infection in a human subject or in a non-human animal, e.g. a non-human animal as hereinabove defined.

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.

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.

Description of the figures

Figure 1A - D. The aligned amino acid sequences of the VHH clones that inhibit urease activity (see Tables 6, 13 and 14), including the delineation of their resp. FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4 regions, and amino acid positioning in Chotia, IMGT and Kabat numberings. Asparagine residues forming a potential N-glycosylation site are boxed.

Figure 2. SDS-PAGE analysis of the yeast produced VHHs (see Table 6). SDS-PAGE was done using 4-12% NuPAGE Bis Tris gels in MES running buffer under reducing conditions (ThermoFisher Scientific). Gels were stained with SimplyBlue Safestain (ThermoFisher Scientific). A low molecular weight marker (LMWM) from GE Healthcare was included; the sized of these proteins are indicated at the right. CDR3 groups of VHHs are indicated on top. Samples deglycosylated by endoglycosidase H treatment are indicated by DG. The position of endoglycosidase H and glycosylated VHH is indicated by arrows.

Figure 3. Binding of VHHs to a peptide representing the FLAP region of Jack Bean urease.

Unlabelled VHHs were coated at 5 pg/ml and detected by incubation in buffer containing 0.5 pg/ml streptavidin-horse radish peroxidase and 0.5 pg/ml biotin-Jack Bean urease-FLAP peptide. Bound horse radish peroxidase was detected by TMB staining. DG indicates that VHHs were deglycosylated by treatment with endoglycosidase H. Figure 4A-D. SDS-PAGE analysis of proteolytic stability of 4 urease binding VHHs U49F, U60, U64 and U61 F. All VHHs were expressed as fusion to a long hinge and his6 tag. The different panels each represent a different VHH. The CDR3 group of the VHH is indicated between parenthesis. VHHs were digested with various proteases or mock incubated in pepsin-buffer or trypsin-buffer as indicated. The position of low molecular weight markers (LMWM) as well as bands representing proteases, VHHs or N-glycosylated U61 F is indicated.

Figure 5. Binding of VHHs to Klebsiella aerogenes urease, Jack Bean urease from Sigma or Helicobacter pylori urease. ELISA plates coated with ureases at 5 pg/ml were subseguently incubated with tenfold diluted E. co// supernatants containing VHHs. Clones 156-4G8 and 156-8F10 are shown for negative control. Bound VHHs were detected with GAL-PO and TMB staining.

Absorbance values at 450 nm were measured.

Examples

1 . Llama immunization and phage display library generation

Immunization of Llamas (Lama glama) (no.’s L9275 and L9276) was done under animal ethical protocol license 2019.D-0004.010, using antigens as listed in Table 1 .

MCR and Urease from Protein Specialists, ENZ-277, were dissolved in sterile water, H. pylori urease was supplied as liguid in PBS and both types of jack bean urease were dissolved in PBS. The concentrations of antigens were checked by Bradford assay. The llama immunization schedule is described in Table 2. The E. co//-produced proteins were depleted of endotoxins prior to immunization using Pierce cat no. 88274 0.5 ml High Capacity Endotoxin Spin Columns. They were then pooled with the other antigens in PBS as described in Table 1 , buffer changed to 20 mM Hepes pH 6.5 1 300 mM NaCI buffer, diluted to 2 ml of this buffer and mixed with 2 ml Gerbu FAMA adjuvant for a single intramuscular injection of one llama (Table 2).

Serum samples were collected at 0, 28 and 49 days post primary immunization (DPI) and

150-ml heparinized blood samples were collected at 28 and 49 DPI. These large heparinized blood samples were used for isolation of peripheral blood lymphocytes (PBLs). Total RNA was extracted from PBLs using the RNeasy Maxi kit (Qiagen) and used for preparation of cDNA using dT18 priming and Superscript III reverse transcriptase (Thermo Fisher Scientific, Rockford, IL, USA). Three PCRs specific for VHHs were performed using primers BOLI 192 (5’-AACAGTTAAG CTTCCGCTTG CGGCCGCTAC TTCATTCGTT CCTGAGGAGA CGGT), lam07 (5’- AACAGTTAAG CTTCCGCTTG CGGCCGCGGA GCTGGGGTCT TCGCTGTGGT GCG), and a mixture of primers lam08 (5’-AACAGTTAAG CTTCCGCTTG CGGCCGCTGG TTGTGGTTTT GGTGTCTTGG GTT) and BOLI401 (5’-AACAGTTAAG CTTCCGCTTG CGGCCGCTGG TTGTGGTTGT GGTATCTTGG GTT), all three in combination with primer VH2B (Frenken et al., 2000). The resulting PCR fragments were digested with Pstl and Notl and inserted into phage display plasmid pRL144 (Harmsen et al., 2005). The ligations were used to transform E. coli TG1 cells (Lucigen, Middleton, Wl, USA) by electroporation, resulting in twelve libraries (designated PAL1204 to pAL1215; Table 3).

Llama sera were titrated in 3-fold dilution series starting with 100-fold dilution in ELISA on ELISA plates coated with 1 pg/ml JBUS, KAU or HPUCE (see Table 4 for abbreviations) and subsequently bound llama antibodies were detected with goat anti-llama IgG PO conjugate (GAL- PO; Bethyl laboratories, A160-1 OOP) or with an anti-VHH PO-conjugate (aVHH-PO; Genscript, A01861-200). Both llamas had essentially similar responses (data not shown). Good immune responses were seen using GAL-PO against JBUS and HPUCE, but a much weaker response was found against KAU. Titres were already high at 28 DPI. Only for JBUS the heavy-chain antibody response was measured using aVHH-PO. Titres were lower as compared to GAL-PO ELISAs. Furthermore, day 28 sera had clearly lower anti-JBUS responses as compared to day 49, indicating that the day 49 phage display libraries of VHHs are also better. The lower response against KAU could be due to LPS depletion of KAU before immunization causing also KAU depletion. 2. Ureases

The ureases used are summarized in Table 4.

The urease from K. aerogenes has mutation H219Q in the alpha subunit. H219Q urease has ca. 100-fold higher catalytic efficiency resulting from a 10-fold lower Km and a 10-fold higher kcat (Pearson et al., 2000), contrary to mutant H219A (Jabri and Karplus, 1996) that has a lower catalytic efficiency (Pearson et al., 2000). The 3D-structures of authentic and H219Q mutant urease are resolved (Jabri et al., 1995; Pearson et al., 2000); PDB code 1 EJT for H219Q. H219Q urease has a pH optimum that is shifted to pH 6.2 (Pearson et al., 2000).

3. Urease assays

Ureases catalyse the hydrolysis of urea to carbamic acid and NH3. Carbamic acid spontaneously decomposes in solution to yield carbonic acid and an additional NH3. So the overall reaction is: CO(NH2)2 + H2O --> CO2 + 2 NH3. The increased ammonia concentration gives a rise in pH. The enzyme has a pH optimum of 7.5 (wikipedia). The urease enzyme assays either are based on detection of formation of ammonia or on detection of the rise in pH using phenol red. We used the Phenol red assay as well as the Berthelot assay for measuring ammonia.

3. 1 Phenol red assay

Phenol Red colour changes from yellow to red from pH 6.8 to 8.2. Red is measured at 562 nm. Briefly the assay is as follows. Urease assays were done in 96-well format using a final volume of 150 pl per well at 37°C. Ureases were diluted into 75 pl 20 mM Hepes pH 6.5 and then mixed with 75 pl 20 mM Hepes pH 6.5 containing 0.5 M urea and 0.02% phenol red (Urea/Fenol red buffer; final concentration is 250 mM urea and 0.01 % phenol red). A kinetic read was then done at 37°C and 562 nm for either 3 hours with 0.5 hour intervals or for 6 hours with 1 hour interval. For measuring inhibition of urease activity by antibodies or VHHs these antibodies were mixed with urease in 75 pl 20 mM Hepes pH 6.5 and preincubated at 37°C for 1 h before addition of 75 pl Urea/Phenol red buffer and preforming a kinetic read as described above. The percentage inhibition was then calculated as follows: 100 x (1 - (absorbance with antibody-absorbance without urease)/(absorbance without antibody-absorbance without urease)).

3.2 Berthelot’s assay

Berthelot's reagent is an alkaline solution of phenol and hypochlorite, used in analytical chemistry (Weatherburn, 1967). Ammonia reacts with Berthelot's reagent to form a blue product which can be detected spectrophotometrically. The reagent can also be used to detect the formation of ammonia by conversion of urea due to urease enzyme activity. We used a miniaturized version suitable for 96-well plates based on a published protocol (Richmond and Yep, 2019). The protocol is briefly as follows. Urease assays were done in 96-well format PCR plates using a final volume of 30 pl per well at 37°C. Ureases were diluted into 15 pl 20 mM phosphate buffer pH 7.5 and then mixed with 15 pl 20 mM phosphate buffer pH 7.5 containing 0.6 M urea and 40 mM EDTA (2x UPE buffer; final concentration is 300 mM urea and 20 mM EDTA). Samples were then incubated for 1 h to overnight at 37°C. For measuring inhibition of urease activity by antibodies or VHHs these antibodies were mixed with urease in 15 pl 20 mM phosphate buffer pH 7.5 and preincubated at 37°C for 1 h before addition of 15 pl 2xUPE buffer and continuing incubation at 37°C as described above. Then 10 pl samples were transferred to a 96-well ELISA plate and the following was added: 20 pl Berthelot solution A (1 % phenol; 0.005% sodium nitroprusside), 20 pl Berthelot solution B (0.2% sodium hypochlorite in alkali solution), 100 pl water. After mixing at 800 rpm for 15 min the absorbance at 570 nm was measured. The percentage inhibition was then calculated as follows: 100 x (1 - (absorbance with antibody-absorbance without urease)/(absorbance without antibodyabsorbance without urease)).

4. Phage display selection of urease binding VHHs

The phage display selections were done using both D28 and D49 libraries and using directly coated KAU, JBUS, HPUCE and PMU ureases at concentration of 1 or 5 pg/ml. Two panning rounds were done and phage selected the first round on KAU, JBUS or HPUCE were selected the second round on all 3 of these antigens, thus both the same antigen as the first round, as well as two other antigens. Phage selected on PMU were only selected on PMU the second round since this antigen was not used for immunization of llamas. After two selection rounds the repertoires that gave sufficiently high phage ELISA signals were plated for single colonies. In total six 96-well masterplates (145-1 , 145-2, 145-3, 145-4, 146-1 , 146-2) were inoculated with single colonies and used for induction of production of soluble VHHs.

The E. coll supernatants were tested at 20-fold dilution in ELISA on directly coated 2 pg/ml HPUB, JBUS, KAU and PMU using mouse monoclonal antibody clone 9E10 conjugated to HRP (Roche Applied Science) for detection of bound VHHs containing a myc tag. A selection of E. coll supernatants was also tested in an urease inhibition assay. The assay was done at 37°C for 135 min using 20 mM phosphate pH 7.0 buffer and 25 pl E. coll supernatant, using also E. coll supernatant for control incubations with and without urease. Five HPU binders were tested with 20 pg/ml HPUCE, but the A562 obtained with urease was barely above the signal without urease and as a result the assay was not valid and inconclusive with respect to enzyme inhibition. The same applies to analysis of PMU/KAU binders using 5 pg/ml KAU. However, analysis of 2 96-well masterplates with JBU binders in phenol red assay using 2 pg/ml JBUS showed conclusive results: A562 with urease in E. coli sup was about 3.2 whereas A562 with E. coli sup only was about 2.7. Several VHH clones were found to decrease the absorbance value, sometimes even below 2.7, resulting in percentages inhibition above 100%.

Based on ELISA signals on JBUS, HPUCE, KAU and PMU and inhibition of JBUS in Phenol Red assay 187 clones were selected for a novel induction of soluble VHH, among which 82 clones were selected for sequencing. They were induced in masterplates 147-1 to 147-4 (four plates) and contained also many VHHs against other (plant viral) targets that were suitable negative controls in Phenol Red assay. A novel Phenol Red assay was done using these E. coli supernatants using 20 mM Hepes pH 6.5 buffer, 25 pl E. coli supernatant and control incubations with and without urease both without E. coli supernatant. Many urease negative clones were also tested to ascertain that inhibition found was due to specific binding to urease rather than an aspecific effect. 0.33 pg/ml JBUS and 2.5 pg/ml PMU were used. In addition to that, one plate was also tested with 0.065 pg/ml JBUS and only 5 pg/ml E. coli supernatant. In this plate, the same clones were found to inhibit as when using fivefold higher urease and E. coli sup concentrations. This indicates that the assay was valid. Furthermore, we never saw inhibition of urease activity by urease negative VHH clones. The data of these assays were mostly consistent with the earlier assays.

5. Sequence analysis

For the determination of the VHH sequence DNA fragments for sequence analysis were obtained by PCR on E. co//TG1 cells using Phusion PCR mix (ThermoFisher Scientific) with primers MPE25 (5’-TTTCTGTATGGGGTTTTGCTA) and MPE26 (5’-GGATAACAATTTCACACAGGA). Sequence analysis was done using the BigDye Terminator v 1.1 Cycle Sequencing Kit and an automated SeqStudio Genetic Analyzer (Applied Biosystems by Life Technologies). Purified PCR fragment was used as template in combination with primers MPE25 and RevSeq (5’- TCACACAGGAAACAGCTATGAC). All sequence interpretation was done based on the translated VHH sequence. The Pstl site used for VHH cloning overlaps with amino acids 4 and 5 of the mature VHH. Therefore, the sequence QVQ (amino acids 1-3) that is encoded by the phage display vector used was appended to the VHH N-terminus. VHHs were aligned according to IMGT numbering system (Lefranc et al., 2003) of the mature VHH encoding region, ending at sequence VTVSS. VHHs were classified into subfamilies as done earlier (Harmsen et al., 2000). Subfamily C indicates conventional like VHH, lacking the FR2 residues that are typical of VHHs. Such VHHs are often produced at lower levels. Subfamily 1 , 2 and 3 designated VHHs indicate three genuine VHH subfamilies. Subfamily X indicates VHHs that are not classifiable. VHHs were also classified into CDR3 groups based on having identical CDR3 length and at least 65% sequence identity in CDR3. VHH sequences were also inspected for the presence of potential N-glycosylation sites (Asn-X- Ser/Thr, where X is any amino acid except Pro). Clones selected for yeast expression preferentially lacked such N-glycosylation sites. 6. Production of VHHs in yeast

On the basis of preliminary screening for binding to and inhibition of ureases, 24 of the 70 unique VHHs were selected for production in yeast (see Table 5). Yeast production of VHHs was done as described previously (Harmsen et al., 2022) using plasmid pRL188. Pstl-BstEII inserts were codon optimized for yeast expression by Genscript corporation preventing internal Pstl, BstEII, Sacl and Hindi II sites. Expression was done at 0.5 liter scale using Saccharomyces cerevisiae strain SU51 and VHHs were purified by IMAC and buffer changed to PBS. The protein concentration was determined by Bradford protein assay using bovine IgG standard and VHHs were biotinylated using sulfo-NHS-LC biotin (Pierce) at 1 :5 weight ratio of biotin :protein . Based on the yield of VHH from the 0.5 liter culture the yeast production level was determined (Table 5). The clones from CDR3 groups 18 and 19, that are interesting because they inhibit both JBU and PMU, are all produced reasonably well (8 to 35 mg/L).

The aligned amino acid sequences of the yeast-expressed VHH clones in Table 5, with SEQ

ID NO.s: 1 - 11 , are set forth in Figures 1 A-D, including delineations of resp. the FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4 regions and their positions in Chothia, IMGT and Kabat numberings.

The purified unlabeled VHHs were also analyzed on SDS-PAGE, aiming for 1 pg VHH per lane (Figure 2). The VHHs having potential N-glycosylation sites were analyzed on SDS-PAGE both untreated and deglycosylated using endoglycosidase H (DG). Among the VHHs with glycosylation sites only U61 F was clearly glycosylated as indicated by a broad band in the high molecular weight region that disappears after deglycosylation, while the intensity of the VHH migrating at about 25 kDa slightly increases. Since the band in the high molecular weight region is about the same intensity as the band migrating at 25 kDa we estimate this VHH to be glycosylated for about 50%. The VHH U60F contains an N-glycosylation site at the same position and appears to be glycosylated for about 10% since a faint band in the high molecular weight region is visible. None of the other 3 VHHs with N-glycosylation sites appear glycosylated. The U4F VHH is slightly degraded, as indicated by the smear in the region below 14.4 kDa. None of the other VHHs appear degraded. The clones from CDR3 groups 18 and 19 that are most interesting have quite similar band intensities that are average intensities.

7. Titration of VHHs in ELISAs usinq different ureases

ELISAs were done to characterize the 24 VHHs that were selected for production in yeast (see Table 6). Part of the purified batches of 2 VHHs, U60F and U61 F, were also deglycosylated by endoglycosidase H treatment. These are indicated by the suffix DG in Table 6. In addition 3 yeast-produced control VHHs that were earlier reported to bind HPU were also tested: U1 F (Chen and Zihao, 2018), HMR23F (Hoseinpoor et al., 2014), and UC3L (Fouladi et al., 2019). The latter is actually a human single domain antibody derived from the light chain of a human antibody.

Furthermore, negative controls included the M8F VHH (Harmsen et al., 2007) binding to foot- and-mouth disease virus (FMDV) and ELISA buffer only.

ELISAs were performed essentially as described earlier in Harmsen et al., 2022.

The following ELISA setups were used for immobilization of ureases to plates:

• Coating of 2 pg/ml Jack Bean Urease from Sigma (JBUS), U4002-100KU.

• Coating of 2 pg/ml Helicobacter pylori Urease from Biorbyt (HPUB), orb98873.

• Coating of 2 pg/ml Klebsiella aerogenes Urease (KAU), Protein Specialists, ENZ-277.

• Coating of 2 pg/ml Proteus mirabilis Urease (PMU), ThermoFisher Scientific, 15905538.

• Coating with 0.5 pg/ml unlabeled U63F VHH and subsequent capture of 2 pg/ml JBUS

• Coating with 0.5 pg/ml unlabeled U8F VHH and subsequent capture of 2 pg/ml KAU

In all these 6 ELISAs the above mentioned VHHs, in biotinylated form, were titrated from 1 pg/ml over 8 wells in 3-fold dilution series, using ELISA buffer with 1 % milk and 0.05% Tween 20, as earlier described (Harmsen et al., 2022). Bound biotinylated VHHs were detected using a streptavidin - horse radish peroxidase conjugate. Peroxidase was detected by TMB staining and stopped using sulfuric acid. The absorbance at 450 nm (A450) was then measured. The absorbance values at 1 pg/ml VHH are summarized in Table 6. From these data we conclude the following.

None of the 3 positive control VHHs bind to any of the ureases.

Deglycosylation of U60F and U61 F does not result in increased A450 values using any of the ELISA setups. It possibly results in slightly decreased A450 values, which may be an artefact due to the incubation at 37°C. Further conclusions are based on untreated forms of U60F and U61 F.

PMU and KAU give highly comparable results, suggesting that PMU is actually the same antigen as KAU. We therefore focus on KAU for further conclusions below.

Capture of JBUS with U63F does not improve ELISA signals of any of the VHHs but gives similar A450 values as compared to directly coated JBUS. Similarly, capture of KAU with U8F results in a similar pattern of ELISA signals as compared to directly coated KAU. However, capture of KAU with U8F results in systematically lower A450 values as compared to directly coated KAU. None of the VHHs showed substantially increased A450 values due to use of a VHH capture for urease immobilization. We therefore focus on directly coated JBU and KAU for further conclusions below.

U3F, U4F, U5F and U54F bind HPU. U63F, U25F and U64F (CDR3 group 8), U33F (CDR3 group 15), and U30F, U36F and U48F (CDR3 group 18) bind specifically to JBUS, although U64F shows a low A450 value (0.3535) on KAU. U19F (CDR3 group 9) and U24F (CDR3 group 10) bind specifically to KAU, although U19F shows a low A450 value (0.1858) on JBUS. The remaining 9 VHHs belonging to CDR3 groups 5, 6, 7, 19 and 20 are all positive on both JBUS and KAU.

The further titrations of these VHHs on directly coated KAU and JBUS was also used to calculate the optimal biotin-VHH concentration to perform competition ELISAs with unlabeled VHHs for mapping binding to independent antigenic sites.

8. Binding of VHHs the FLAP region of Jack Bean urease

We ordered a biotinylated peptide representing the FLAP region of Jack Bean urease. This peptide is often bound by urease inhibiting antibodies. It has been used previously for isolation of inhibiting antibodies (Ardekani et al., 2013; Chen and Zihao, 2018; Fouladi et al., 2019; Hoseinpoor et al., 2014). The peptide is thought to be a flexible region that covers the urease active site. A biotinylated peptide termed biotin-JBU-FLAP representing amino acid residues EHLDMLMVCHHLDREIPEDLAFAHSRIRKKTIA (SEQ ID NO: 47) of Jack Bean urease was ordered at Genscript Corporation. An ELISA was performed using coating of 5 pg/ml unlabeled VHH and subseguent incubation, in a single step, with a mixture of 0.5 pg/ml biotin-JBU-FLAP and 0.5 pg/ml streptavidin-HRPO conjugate in EBTM. This single step incubation facilitates detection of low affinity interactions. Only U63F was found to bind this peptide, but with a high A450 value of >2 (Figure 3).

9. Mapping of antigenic sites

The antigenic sites of the VHHs binding to JBUS and all VHHs binding to KAU were mapped by competition ELISAs. Essentially the same ELISA setup was used as described above fortitration of VHHs. Plates were coated with 2 pg/ml JBUS or KAU and then blocked by incubation with 90 pl per well of 5 pg/ml unlabeled VHH. After 30 min incubation we added 10 pl of the second biotin- labelled VHH at a concentration suitable for resulting in about 80% of the maximal A450 value, as determined in the ELISA above. This competition step was continued for 45 min at RT. Then plates were washed and bound biotin-VHH was detected using streptavidin-HRPO conjugate. Bound horse radish peroxidase was detected using TMB staining, that was stopped using sulfuric acid. Then A450 values were measured. Control incubations were done without antigen (Ag) coating to determine background A450 values. The % inhibition of antigen binding due to a competing VHH was calculated as 100-100* ([A450 with competing VHH] - [A450 without Ag coating]) I ([A450 without competing VHH] - [A450 without Ag coating]). The results are summarized in Tables 7A and 7B.

The VHHs from CDR3 groups 5, 6 and 7 (Tables 7A and 7B) and in case of JBUS also 15 (Tabel 7A), and in case of KAU also group 9 (Table 7B) appear to bind a single antigenic site or partly overlapping antigenic sites. VHHs from CDR3 groups 8, 18, 19, 20 and 25 all bind a single site on JBUS as judged from inhibition by unlabeled CDR3 group 8 or 18 VHHs (Table 7A). However, inhibition by unlabeled CDR3 group 19, 20 and 25 VHHs was not observed for biotinylated CDR3 group 8 and 18 VHHs and much weaker for biotinylated CDR3 group 19, 20 and 25 VHHs (Table 7A). This is most likely due to differences in affinity of these VHHs since CDR3 groups 8 and 18 VHHs also performed better when titrated in ELISA, resulting in a lower biotinylated VHH concentration suitable for use in competition ELISAs (Table 7A). VHHs from CDR3 groups 19, 20 and 25 also form a separate antigenic site using KAU (Table 7B). The single VHH from CDR3 group 8 that shows some cross reaction with KAU, U64F, shows some non-reciprocal competition with VHHs from CDR3 groups 19, 20 and 25, but in this case the unlabeled U64F does not inhibit biotinylated VHHs from CDR3 groups 19, 20 and 25 while biotinylated U64F is weakly inhibited by 4 out of 5 VHHs from CDR3 groups 19, 20 and 25 (Table 7B). This probably again reflects differences in affinity for KAU, which is also suggested by the low absorbance value of U64F on KAU as compared to VHHs from CDR3 groups 19, 20 and 25 (Table 6).

The single VHH from CDR3 group 10, U24F, appears to bind the same site as U64F from CDR3 group 8 (Table 7B). This is surprising since U24F is specific for KAU while U64F shows much stronger binding to JBUS.

Taken together, these results suggest that VHHs from CDR3 groups 19, 20 and 25 recognize the same site on urease that is conserved on KAU and JBUS, while VHHs from CDR3 groups 8 and 18 recognize an at least partly overlapping site present on JBUS that is mostly not present on KAU, ignoring the slight binding of KAU by U64F. Since U63F, which is from CDR3 group 8, binds a peptide representing the FLAP region of JBUS the other VHHs of CDR3 groups 8 and 18 presumably also bind this peptide, but not in sufficient affinity to result in a positive ELISA signal, possibly because other regions of urease also form an important part of the epitope. By inference, the site recognized by VHHs from CDR3 group 19, 20 and 25 most likely also is close to the FLAP peptide. 10. Inhibition of urease activity

Inhibition of urease activity was measured using the Fenol Red assay. Briefly, VHHs were preincubated with urease in 75 pl 20 mM Hepes pH 6.5 buffer at 37°C for 1 h in a polystyrene ELISA plate. Control incubations were done with urease without VHH and with buffer only. Then, 75 pl of 20 mM Hepes containing 0.02% Fenol Red and 0.5 M urea was added, resulting in 0.01 % Fenol Red and 250 mM urea final concentrations. Incubation was continued at 37°C using a kinetic read at 562 nm using the SoftMax Pro reader for 4 h with 100 min interval or 50 min interval. The % inhibition of urease activity due to a VHH was calculated as 100-100* ([A562 with urease and with competing VHH] - [A562 without urease]) I ([A562 with urease but without VHH] - [A562 without urease]). The VHH concentration resulting in half maximal inhibition was defined as the 50% inhibitory concentration (IC50). It was calculated for individual VHHs by making a 4-parameter curve fit of plots of percentage inhibition versus VHH concentration and interpolating the IC50. The results are presented for inhibition of Jack bean urease by 10 pg/ml VHHs in Table 8, for inhibition of K. aerogenes urease and Jack urease by dilution series of selected VHHs in Table 9 and 10, respectively, and for inhibition of Jack bean urease by combinations of VHHs in Table 11 .

U60F appears the best inhibiting VHH based on prolonged incubation for 400 min in the phenol red assay (Table 8). Deglycosylation of U60F and U61 F did not improve inhibition of JBUS. The VHHs from CDR3 groups 1 , 2, 3, 4, 5, 6, 7, 9, 10, 15 and 27 do not inhibit Jack bean urease (Table 8). This is consistent with the absence of binding to Jack bean urease in ELISA by VHHs from CDR3 groups 1 , 2, 3, 4, 9, 10 and 27 (Table 6) and the mapping to a single antigenic site of VHHs from CDR3 groups 5, 6, 7 and 15 (Table 7A).

Titration of VHHs in urease inhibition assays using both K. aerogenes urease (Table 9) and Jack bean urease (Table 10) was done with the 11 VHHs from CDR3 groups 8, 18, 19, 20 and 25 that inhibited Jack bean urease at 10 pg/ml VHH concentration (Table 8) as well as 6 VHHs from CDR3 groups 5, 6, 7 and 9 that bind KAU in ELISA (Table 6) and negative control VHH M8F. The 3 VHHs from CDR3 group 8 (U63F, U25F, U64F) bind better to JBUS than KAU in ELISA. They inhibit both JBUS and KAU.

The 3 VHHs from CDR3 group 18 (U30F, U36F and U48F) bind specifically to JBUS in ELISA (Table 6) and show good inhibition of JBUS (Table 10) but not KAU (Table 9). The 3 VHHs from CDR3 group 8 also bind specifically to JBUS in ELISA, although U64F binds with low A450 value (0.3535) to KAU (Table 6). These VHHs however inhibit both JBUS (Table 10) and KAU (Table 9). Consistent with its slightly higher A450 value in ELISA using KAU, U64F shows slightly better inhibition of KAU (lower IC50) as compared to the other 2 VHHs from CDR3 group 8. The 4 VHHs from CDR3 groups 19 and 20 bind to both JBUS and KAU in ELISA (Table 6) and inhibit both KAU and JBUS (Table 9 and 10). In general the IC50 of VHHs was lower using JBUS than KAU, which is partly due to the 2-fold lower urease concentration used in phenol red assays using JBUS as compared to KAU (Tables 9 and 10).

We also analyzed the effect on Jack bean urease inhibition of mixing two VHHs of U64F, U30F, U49F and U61 F, representing CDR3 groups 8, 18, 19 and 20. The mixtures of VHHs had a total VHH concentration that is equal to the VHH concentration of single VHHs. It is evident that mixing VHHs does not improve inhibition as compared to single VHHs (Table 11).

11 . Proteolytic stability of VHHs

For inhibition of urease activity in the gastrointestinal tract after oral application of VHHs the VHHs should be able to resist proteolytic degradation. Several proteases occur in the gastrointestinal tract. Pepsin occurs in the stomach. It has a pH optimum of 2.0 for cleavage. Trypsin and chymotrypsin occur in the small intestine and have a more neutral pH optimum. Protein proteolysis was recently reviewed (Lai et al., 2021). Furthermore, oral application of VHHs and the relevance of proteolytic stability was recently reviewed (Petersson et al., 2023). The proteolytic stability of VHHs is highly variable. Some VHHs are rapidly degraded whereas others can have high proteolytic stability. Such differences in stability are dependent on the VHH sequence and 3D structure. As a result various studies have been done to improve proteolytic stability of VHHs by random mutagenesis approaches or rational protein engineering (Harmsen et al., 2006; Hussack et al., 2011 ; Hussack et al., 2014; Rutten et al., 2012). Furthermore, panels of VHH clones can be screened for their susceptibility to proteolytic degradation, to identify the more stable VHHs (Harmsen et al., 2006; Maffey et al., 2016).

Therefore, we assessed the proteolytic stability of the eleven VHHs that inhibit JBUS activity (U63F, U25F, U64F, U30F, U36F, U48F, U49F, U58F, U60F, U61 F and U42F) by both SDS-PAGE and analysis of urease binding in ELISA. Protease assays were done as earlier described (Harmsen et al., 2006) by preincubation of yeast-produced VHHs with different concentrations of bovine pancreatic trypsin (Type 1 ; Sigma Aldrich cat. no. T8003), bovine pancreatic chymotrypsin (type II Sigma Aldrich cat. no. C4129) or porcine pepsin (Roche, cat. no. 10108057001). Incubations with pepsin were performed in 10 mM HCI (pH 2; pepsin buffer). Incubations with trypsin or chymotrypsin were performed in 1 mM Tris. Cl (pH 8.0), 20 mM CaCh (chymotrypsin buffer). Pepsin was used at concentrations of 0.1 , 0.01 , 0.001 and 0.0001 mg/ml. Trypsin and chymotrypsin were used at concentrations of 0.1 , 0.01 and 0.001 mg/ml. For quantitative measurements a 1-pg amount of purified yeast-produced VHH was incubated in 10 pl of protease solution in either pepsin- or chymotrypsin buffer for 1 h at 37°C. For subsequent SDS-PAGE analysis (NuPage gels; Thermo Fisher Scientific) the reaction was stopped by addition of a premix of 1 .6 pl NuPage reducing agent, 4 pl 4xNuPage loading buffer and 1 pl 1 M Tris. Cl pH 8.0. SDS-PAGE analysis was done by heating samples as prepared above for 10 min at 70°C. The samples were then analyzed using 4-12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) and MES running buffer (Invitrogen), stained using Simply Blue SafeStain (Thermo Fisher Scientific). Gels were visually inspected for band intensity and protein construct integrity.

For subsequent ELISA analysis the samples were tenfold diluted in ELISA buffer containing 1 % skimmed milk, 0.05% Tween-20, 100 mM Tris. Cl pH 8.5 and cOmPlete protease inhibitor (Roche Applied Science) and immediately analysed by ELISA. The amount of functional VHH was then determined by antigen-specific ELISA as follows. Polystyrene 96-well plates (Greiner, Solingen, Germany) were coated with 5 pg/ml Jack Bean urease in 0.05 M carbonate/ bicarbonate buffer, pH 9.6. Residual sites were blocked for 1 hour with EBTM containing protease inhibitors. After incubation with serial threefold dilutions of VHHs, bound VHHs were detected with 5000-fold diluted peroxidase-conjugated goat anti-llama immunoglobulins (Bethyl Laboratories) and staining with 3, 3', 5, 5' tetramethylbenzidine. The VHH concentrations of proteolytically treated samples were then interpolated in a standard curve of concentrations of VHHs in pepsin or chymotrypsin buffer without protease versus absorbance at 450 nm. The percentage functional VHH remaining after proteolysis as compared to control incubations without protease was then calculated.

Due to a too low ELISA signal (A450<0.3) of the untreated control VHH the percentage residual VHH could not be determined for U58F and U61 F. In case of U61 F this could partly be due to 50% of the VHH being N-glycosylated (Fig. 4D), which could hinder binding of the polyclonal HRPO conjugate used for VHH detection. In case of U42F the maximal A450 value was also very low (0.187), but results were suitable for curve fitting, contrary to U58F and U61 F. For all other urease binding VHHs the percentage residual VHH could be calculated based on ELISA (Table 12). In most cases increasing protease concentrations resulted in decreasing percentage residual VHH.

The digested VHH samples were subsequently analyzed by SDS-PAGE (Figure 4). Consistent with the predicted molecular weight, pepsin migrates at about 40 kDa, chymotrypsin migrates at 25 kDa and trypsin at 24 kDa. All three proteases form sharp bands. Pepsin does not show auto degradation bands. Trypsin, however, shows 4-6 sharp bands in lanes 6 of each panel, that are most likely trypsin autoproteolysis products. Similarly, chymotrypsin shows 4-5 discrete bands in lanes 9 of each panel that appear mostly identical and thus probably represent autoproteolysis of chymotrypsin. The urease binding VHHs migrate at about 26 kDa, often overlapping the trypsin and chymotrypsin bands. The VHH bands are much more diffuse, due to heterogeneous O-glycosylation in the long hinge region that is C-terminal of the VHH (Harmsen and Fijten, 2012).

ELISA data are generally consistent with SDS-PAGE for pepsin. The intensity of the diffuse band corresponding to the VHH often correlates well with % VHH measured by ELISA. An exception is U42F (data not shown), that does not show a VHH band at 0.001 or 0.01 mg/ml pepsin, while having 42% and 27% residual VHH in ELISA. Often a diffuse band migrating at about 14-17 kDa appears when using higher pepsin concentrations. This is probably a degradation product that is unable to bind urease. Possibly this VHH breakdown product is able to bind urease in case of U42F.

Limited trypsin digestion using 0.01 mg/ml trypsin often results in a discrete VHH breakdown product migrating at about 16-17 kDa. The appearance of this 16-17 kDa band correlates with the disappearance of the about 26 kDa band representing intact VHH. Most often this 16-17 kDa band is still present at 0.1 mg/ml trypsin, but with lower intensity, and disappears using 1 mg/ml trypsin. This is most likely a VHH breakdown product that is C-terminally truncated since the band is discrete, suggesting the VHH is not O-glycosylated. A 14 kDa trypsin degradation product that appears to be stable at all trypsin concentrations is seen with U25F (not shown) and U64F (Figure 4C), which is confirmed by ELISA. Possibly this product shows lower reactivity to the polyclonal antibody used for VHH detection in ELISA, explaining the lower percentage residual VHH found (Table 12), i.e. the percentage residual VHH as measured by ELISA may be an underestimation of the actual amount of residual VHH capable of antigen binding. A similar 16-17 kDa trypsin breakdown product is also seen for the further 8 urease binding VHHs. In case of the 3 CDR3 group 19 VHHs the breakdown product appears to migrate a bit lower, at 16-kDa and ELISA data indicate that these 3 VHHs are highly sensitive to trypsin, suggesting that this 16 kDa product is not capable of antigen binding.

Chymotrypsin digestion often results in a breakdown product slightly below the 14.4 kDa marker band already at the lowest protease concentration (0.01 mg/ml) used. Only in case of U58F and U60F of CDR3 group 19 this product is not visible. At 0.1 or 1 mg/ml chymotrypsin the 14-kDa degradation product is mostly absent for the 11 VHHs. Using chymotrypsin the intact VHH band is still visible at 0.01 mg/ml chymotrypsin, although sometimes at slightly reduces intensity, but disappeared at 0.1 (and 1) mg/ml chymotrypsin for all 1 1 VHHs. As was also observed for trypsin, the stability observed in ELISA is often higher as compared to SDS-PAGE at higher chymotrypsin concentrations. This again suggests that some chymotrypsin degradation products still retain antigen binding capacity.

Both ELISA (Table 12) and SDS-PAGE analysis (Figure 4A,C), show that U64F and U49F are relatively proteolytically stable VHHs. U49F and U64F show the highest pepsin resistance, with 13-30% residual VHH at 0.01 mg/ml pepsin (Table 12). U64F also shows the highest resistance against chymotrypsin digestion, with 36% residual VHH at 1 mg/ml chymotrypsin (Table 12), and is one of the clones with 14% residual VHH in ELISA at 1 mg/ml trypsin (Table 12). U49F is clearly less resistant against trypsin and chymotrypsin than U64F, both based on ELISA and SDS-PAGE. U42F also appears relatively stable in ELISA (Table 12) but the pepsin resistance is not confirmed in SDS-PAGE (not shown). U60F shows reasonable resistance against pepsin, but low resistance against trypsin and chymotrypsin (Table 12 and Figure 4B). U61 F also appears reasonably resistant against proteases, especially pepsin, but trypsin resistance appears lower. U61 F stability is only based on SDS-PAGE analysis (Figure 4D) by comparison of the bands of unglycosylated VHH migrating at about 26 kDa.

12. Novel llama immunization with Klebsiella aeroqenes urease

Two male llamas numbered 9282 and 9283 were immunized with Klebsiella aerogenes urease (KAU) from Protein specialists (Table 1) under animal ethical protocol license 2019. D- 0004.014. The immunization procedure was highly similar as described in example 1 , using 50 pg urease per immunization per llama and Gerbu FAMA adjuvant. However, we did not do endotoxin removal prior to immunization since LPS depletion could also result in KAU depletion. The immunization scheme as described in Table 2 was used, with start date of 9 May 2023. Similar as described in example 1 , we constructed phage display libraries from PBLs collected at 28 and 49 DPI, using back primers lam07, Iam08/BOLI401 and BOLI192 (example 1). Insertion of Pstl-Notl digested PCR fragments in vector pRL144 (Harmsen et al., 2005) resulted in twelve libraries, designated pAL1260-pAL1271 . 13. Selection of proteolytically stable KAU inhibiting VHHs

The phage display selection of novel KAU inhibiting VHHs was done using 28 DPI and 49 DPI libraries separately and using directly coated KAU and JBUS at a concentration of 1 or 5 pg/ml. Several variations were done on the panning procedure. Heating of phage repertoires was earlier used to select for recombinant antibodies with high conformational stability (Jespers et al., 2004; Jung et al., 1999). Some phages were therefore heated for 1 h at 80°C to select for stable VHHs. Most phage display selections were done using PBS of pH 7.2 containing 1 % milk and 0.05% Tween-20 (PBSTM). However, some selections were done using PBSTM of pH 6.5 to mimic the pH of the intestine. Furthermore, bound phage were recovered after panning by incubation in 100 mM triethylamine (TEA, Sigma Aldrich) for 10 minutes followed by instant neutralization with 50 pl 1 M Tris. Cl, pH 7.5. Sometimes such TEA elution was followed by incubation in 1 mg/ml trypsin in PBS (trypsin elution). A second panning round was done on either the same antigen as the first round or on the other urease type. The latter cross-panning approach aimed to select for VHHs that bind both KAU and JBUS. The panning history of the eight clones that were finally selected is indicated in Table 13.

After two selection rounds the repertoires that gave sufficiently high phage ELISA signals were plated for single colonies. In total ten 96-well masterplates (156-1 to 156-10) were inoculated with single colonies and used for induction of production of soluble VHHs. U60 and U64 were also included as positive controls. For this purpose U64 was inserted into pRL144 by doing a nested PCR on the codon-optimized sequence of U64 in plasmid pRL188 (example 6) using primers BOLI5 (5’- ATTATTTTAGCGTAAAGGATGGGG) and BOLI166 (5’- ATGATGCTTTTGCAAGCCTTC) followed by PCR with BOLI6 (5’- CCTTTTCCTTTTGGCTGGTTTTGC) and BOLI409 (5’- CTAGCGGCCGCTGAGGAGACGGTGACCTGGGTCC). The resulting PCR product was digested with Pstl and Notl and ligated with similarly cut pRL144 plasmid. The U60 VHH in plasmid pRL188 (example 6) was similarly inserted into pRL144GAA, pRL144GAA is a derivative of pRL144 where the proteolytically sensitive enterokinase site (amino acids DDDDK), myc tag (amino acids EQKLISEEDLN) and his6 tag (amino acids HHHHHH) were replaced by a short linker encoding amino acids Gly-Ala-Ala. Vector pRL144GAA thus encodes a Notl cloning site, followed by a 9 base pair region encoding Gly-Ala-Ala and the TAG (amber) stop codon preceding the P3 protein (GCGGCCGCAggggccgcaTAG; Gly-Ala-Ala encoding region in lowercase).

The E. coli supernatants were tested at 10-fold dilution in ELISA on directly coated 5 pg/ml JBUS, KAU and HPU using the HRP-conjugated goat anti-llama immunoglobulin antibody (GAL- PO; Bethyl laboratories) for the detection of bound VHHs. Bound HRP was detected by TMB staining, stopping with sulfuric acid and measurement of absorbance at 450 nm. A relative high percentage of clones bound to KAU with absorbance values above 1 , but very few clones appeared to be binding to JBUS or HPU. The absorbance values of a selection of these clones is presented in Figure 5. Two phenol red assays were performed using 25 pl E. coli supernatant and either 0.66 pg/ml KAU measured for 1050 min or 0.33 pg/ml JBUS measured for 60 min to determine if VHHs inhibit urease activity. The assay was done as described in Examples 3 and 4. However, we did not use negative E. coli supernatant in the control incubations with and without urease. Furthermore, we used Hepes buffer pH 6.5 as described in Example 3. Since negative E. co// supernatant already results in an increase in pH this often causes higher absorbance values in the phenol red assay with E. co// supernatant as compared to the controls with urease, which results in negative inhibition values. Furthermore, this increase in absorbance due to E. coli supernatant causes an underestimation of the percentage inhibition values.

The results of this first Phenol red assay of the eight clones finally selected and a number of controls are shown in Table 14. Negative control VHHs 156-4G8 and 156-8F10 show negative inhibition values below -600 using KAU, which is due to the buffer shift due to E. coli supernatant. Since the control with KAU urease gave only an A562 value of about 0.6 such pH effects of E. coli sup can cause such large negative values. Such negative inhibition values were also observed with JBUS, although only up to -61 , since the control with JBUS urease reached much higher A562 values, up to about 1 .9. None of the novel isolated 8 VHHs inhibited JBUS while the positive control VHHs U60 and U64 showed 70% and 48% JBUS inhibition, respectively (Table 14). Six out of eight selected novel VHHs showed KAU inhibition varying from 27% to 96% (Table 14).

On the basis of KAU inhibition values and binding to KAU and JBUS in ELISA, 53 positive clones were picked for second round of screening along with 4 negative clones, and U60 and U64 controls. The second phenol red inhibition assay was performed with 1 .66 pg/ml of KAU for 244 min since the first assay resulted in relatively low A562 values of the positive control with urease. This indeed resulted in higher A562 values of the buffer control with urease and a concomitant lower values for negative inhibition of several samples with E. coli supernatant (Table 14). This assay confirmed that six of the eight novel VHH clones inhibit KAU urease activity with at least 47% (Table 14).

To assess proteolytic stability of VHHs by ELISA E. coli supernatants were preincubated with different concentrations of bovine pancreatic trypsin (Type 1 ; Sigma Aldrich cat. no. T8003), bovine pancreatic chymotrypsin (type II Sigma Aldrich cat. no. C4129) and porcine pepsin (Roche, cat. no. 10108057001) prior to analysis of urease binding in ELISAs. Proteases were used at 1 and 0.1 mg/ml final concentration in 100 pl final volume consisting of 10 pl E. coli supernatant and 90 pl of either trypsin/chymotrypsin buffer (1 mM Tris HCI, pH 8.0 and 20 mM CaCh) or pepsin buffer (10 mM HCI, pH 2.0). After incubation for 1 hour at 37°C or a mock incubation without protease in trypsin/chymotrypsin buffer the samples were twofold diluted with PBSTM containing 100 mM Tris. Cl pH 8.5 and complete protease inhibitor (Roche; cat. no. 11697498001). They were then analysed by ELISA on 96-well polystyrene plates directly coated with 5 pg/ml of KAU and JBUS, using GAL-PO for detection of bound VHHs, as described above. The ELISA analysis of the proteolytic stability of the eight novel VHH clones selected and a number of controls are indicated in Table 15. Since U64 only binds to JBUS, we presented the ELISA data of JBUS for this positive control VHH whereas the ELISA data obtained on KAU are used for the further 9 VHHs since these showed better binding to KAU (Figure 5). The negative controls 156-4G8 and TG1 showed low absorbance values up to 0.218 while the ten U-clones clearly showed binding in ELISA with absorbance values above 1.0 (Table 15). In case of VHH U109 this binding is not visible in the mock incubation although ELISA values above 1 .5 are seen in the three incubations with 0.1 mg/ml of different proteases (Table 15). This strongly suggests that the low A450 value of 0.0769 of U109 without protease is an artefact. In this large screen most VHH clones had a relatively low proteolytic stability against at least one protease and often several proteases. VHH clone U105 also has such a low proteolytic stability against all three proteases (Table 15), but was selected for further work based on its high KAU inhibition (Table 14). The positive control VHH U60 shows very low trypsin or chymotrypsin stability and only partial pepsin stability. The positive control VHH U64 however, does not show a decrease in absorbance values after pepsin digestion, whereas trypsin and chymotrypsin result in a decrease in absorbance values of 2.8941 to about 0.5 at the highest protease concentration (Table 15). Clones U117, U118, U109, U113 and U112 show high pepsin stability with absorbance values comparable to mock control while trypsin and chymotrypsin stability is often high, especially of U117 and U113 (Table 15). Clones U115 and U119 also show high proteolytic stability against the three proteases, but did not inhibit KAU urease activity (Table 14).

Nineteen VHH clones showing inhibition of KAU urease activity or high proteolytic stability were sequenced using the methods described in Example 5. They all encoded different VHHs and were named U101 to U119. Six VHH clones, U105, U109, U112, U113, U117 and U118 showed inhibition of KAU (Table 14). These six VHH clones could prove useful for the intended application. The VHH clones U115 and U119 did not inhibit KAU (Table 14) and are shown for reference. The protein sequences of the six novel urease inhibiting VHHs are shown in Figure 1A-D. VHH clones U117 and U118 were from the same CDR3 group (28) and differed by only 13 amino acid residues. The further six clones were from separate CDR3 groups numbered 29 to 34. These CDR3 groups 28 to 34 differed from the earlier identified CDR3 groups 1 to 27 (Table 5). Thus, we identified completely novel VHHs. The six novel KAU inhibiting VHHs did not bind JBUS or HPU in ELISA while absorbance values against KAU were clearly above background (Figure 5). The control VHH U60 showed a high absorbance value with KAU and a much lower absorbance value of 0.263 on JBUS while U64 bound JBUS but not KAU urease (Figure 5). Both control VHHs did not bind HPU. None of the eight novel VHHs contained potential N-glycosylation sites. The classification of VHHs into CDR3 groups and VHH subfamilies, as defined earlier (Harmsen et al., 2000), is indicated in Table 13. All six novel KAU urease inhibiting VHHs, U105, U109, U112, U113, U117 and U1 18, belong to VHH subfamilies 1 or 2. Interestingly, U113, U117 and U118 have an He residue at IMGT position 28, instead of the Arg residue that often occurs at this position in VHHs. Since trypsin cleaves C-terminal of Lys or Arg residues (Lai et al., 2021) this could be involved in the high resistance against trypsin digestion, that is especially observed with U113 and U117 (Table 15). Others earlier observed that a rotavirus binding VHH showed improved stability against trypsin by mutation of Arg-28 to Ala (Rutten et al., 2012).

^a Percentage residual VHH could not be determined due to a too low ELISA signal of untreated samples.

^a 156-4G8 and 156-8F10 are control E. coli sups of VHHs that do not bind urease. No inoculation is culture medium that was not inoculated. Without urease and with urease are control incubations without E. coli culture supernatant. ^b The percentage inhibition was calculated for each 96-well plate in an assay separately using four controls without urease and four controls with urease. Since ten plates were used in the first assay the A562 values can give different percentages inhibition dependent on the plate origin of the clone. In the second assay the A562 values and % inhibition correlate better since only one plate was used.

^a 156-4G8 is a control E. coli supernatant of a VHH clone that does not bind urease. TG1 is a control E. coli culture supernatant that does not contain VHH.

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Claims

1 . A composition comprising a first polypeptide comprising a first anti-urease antigen-binding domain that inhibits activity of a plant urease, and a second polypeptide comprising a second anti-urease antigen-binding domain that inhibits activity of a microbial urease, wherein the first and second antigen-binding domains are immunoglobulin single variable domains (ISVDs).

2. A composition according to claim 1 , wherein the plant urease is a Jack Bean urease and wherein the microbial urease is a bacterial urease, wherein preferably, the bacterial urease is a urease from Klebsiella aerogenes.

3. A composition according to claim 1 or 2, wherein at least one of: i) the first polypeptide inhibits at least 20% activity of the plant urease; and, ii) the second polypeptide inhibits at least 20% activity of the microbial urease, preferably when assayed at a twofold molar excess of the antigen-binding domain.

4. A composition according to any one of claim 1 - 3, wherein at least one of the first and second polypeptides do not specifically bind to a urease from Helicobacter pylori.

5. A composition according to any one of claim 1 - 4, wherein: i) the first antigen-binding domain cross-blocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 3, 7, 10 and 9, to the plant urease and/or wherein the first antigen-binding domain is cross-blocked from binding the plant urease by at least one of the VHHs; and ii) the second antigen-binding domain cross-blocks the binding of at least one VHH comprising an amino acid sequence as set forth in SEQ ID NO.’s: 61 - 65, to the microbial urease and/or wherein the second antigen-binding domain is cross-blocked from binding the microbial urease by at least one of the VHHs.

6. A composition according to any one of claim 1 - 5, wherein the first antigen-binding domain is a first ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 12, 13, or 14, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 12, 13, or 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 15, 16, or 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 15, 16, or 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 18, 19, or 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 18, 19, or 20; and, b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 25, 26, or 27, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 25, 26, or 27; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 28, 29, or 30, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 28, 29, or 30; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 31 , 32, or 33, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 31 , 32, or 33; and wherein the second antigen-binding domain is a second ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 66 or 67, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 66 or 67; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 68 or 69 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequences of SEQ ID NO.’s: 68 or 69; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70; d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 71 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 71 ; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 72 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 72; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 73 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 73; e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 74, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 74; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 75 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 75; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 76 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 76; f) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 77, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 77; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 78 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 78; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 79 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.: 79.

7. A composition according to claim 6, wherein the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NOs: 20; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NOs: 25, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 25; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 28, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 28; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 31 , or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 31 ; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 34; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 35, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 35; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 36; and wherein the second ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 66, or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.’s: 66; a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO.’s: 68 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO.’s: 68; and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO.: 70 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 70.

8. A composition according to claim 6 or 7, wherein the first ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: a) an amino acid sequence as set forth in SEQ ID NO.’s: 3; b) an amino acid sequence as set forth in SEQ ID NO.’s: 7 or 9; or c) an amino acid sequence as set forth in SEQ ID NO: 10; and wherein the second ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: c) an amino acid sequence as set forth in SEQ ID NO.’s: 61 or 62; d) an amino acid sequence as set forth in SEQ ID NO: 63; e) an amino acid sequence as set forth in SEQ ID NO: 64; or f) an amino acid sequence as set forth in SEQ ID NO: 65.

9. A composition according to claim 8, wherein the first ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: a) an amino acid sequence as set forth in SEQ ID NO: 3; or, b) an amino acid sequence as set forth in SEQ ID NO: 7; and wherein the second ISVD comprises an amino acid sequence with at least 70, or with increasing preference, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86 sequence identity with: c) an amino acid sequence as set forth in SEQ ID NO: 61 .

10. A composition according to any one of the preceding claims, wherein the composition comprises a bispecific polypeptide wherein the first and second polypeptides are fused in a single polypeptide chain, wherein, optionally the first and second polypeptides are linked through a spacer amino acid sequence.

11. A polypeptide as defined in any one of claims 4 - 10.

12. A polypeptide according to claim 13, 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, 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.

13. A nucleic acid encoding the polypeptide of claim 11 or 12.

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 composition as defined in any one of claims 1 - 10 or a polypeptide as defined in claims 11 or 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 composition as defined in any one of claims 1 - 10 or a polypeptide as defined in claims 11 or 12.

16. Use of a composition as defined in any one of claims 1 - 10, or a polypeptide as defined in claim 11 or 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 - 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.