WO2025132833A1

WO2025132833A1 - Anti trypsin-3 single domain antibody

Info

Publication number: WO2025132833A1
Application number: PCT/EP2024/087466
Authority: WO
Inventors: Céline DERAISON; Nathalie Vergnolle; Aurélien OLICHON
Original assignee: Universite de la Reunion Saint Denis; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de La Reunion; Universite Toulouse III Paul Sabatier; Ecole Nationale Veterinaire de Toulouse ENVT; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Current assignee: Universite de la Reunion Saint Denis; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de La Reunion; Ecole Nationale Veterinaire de Toulouse ENVT; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement; Universite de Toulouse
Priority date: 2023-12-20
Filing date: 2024-12-19
Publication date: 2025-06-26
Anticipated expiration: 2026-06-20

Abstract

The invention relates to generation, and characterisation of single domain antibodies targeted against Trypsin-3 with high specificity, obtained by subtractive phage display from a naïve synthetic library. In the present invention, inventors disclosed and generated single domain antibody (sdAb) targeting specifically Trypsin-3 and not the other serine protease of the same family (Trypsin-1 and Trypsin-2). Moreover, using the most promising sdAbs as building blocks, inventors engineered biparatopic single domain antibodies to identify a highly selective binder that tightly inhibit the target protease. This biparatopic single domain antibody specifically inhibits Trypsin-3 activity in malignant growth of PC3 prostate cancer cells and in gut tissue sections from IBS patients, providing a new tool in anti-Trypsin-3 immunotherapies. The invention relates accordingly to single domain antibody that binds specifically Trypsin-3 protein, and not the other serine protease of the same family (Trypsin-1 and Trypsin-2). These specific antibodies can be used for the detection of Trypsin-3 but also for therapy of gut diseases associated with intestinal permeability (ie Irritable Bowel Syndrome (IBS)) including gluten hypersensitivity and for use in treatment of cancer especially cancer associated with Trypsin-3.

Description

ANTI TRYPSIN-3 SINGLE DOMAIN ANTIBODY

FIELD OF THE INVENTION:

The invention relates to single domain antibody that binds specifically Trypsin-3 protein, and not the other serine protease of the same family (Trypsin 1 and Trypsin 2). These specific antibodies can be used for the therapy of gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis and also for the therapy of cancer. In particular, the invention relates to single domain antibody directed specific against Trypsin-3, for use in the treatment of Irritable Bowel Syndrome (IBS) including gluten hypersensitivity and for use in treatment of cancer associated with Trypsin-3.

BACKGROUND OF THE INVENTION:

Proteases are key players in physiology but also in diseases manifestations. Although protease inhibitors have shown some therapeutic success, essentially for HIV infection, only few protease inhibitors have been approved. Indeed, one of the main challenges in developing inhibitors for protease resides in reaching enough selectivity toward the active conformation of the target among more than 550 human proteases (Puente, Sanchez, Overall, & Lopez-Otin, 2003). Indeed, proteases are a large enzymatic family with close homologues that share similarities in their three-dimensional folding and consequently also in their active site conformation. For example, the trypsin-like serine proteases family members share sequence identities in the vicinity of the active site residues and all described inhibitors cross react with several conserved members (Otlewski, Jelen, Zakrzewska, & Oleksy, 2005). However, selective inhibition of serine protease appears crucial since off-target effects can lead to severe disorders (Puente et al., 2003).

Human Trypsin-3 is a unique digestive serine-protease specialized for the degradation of trypsin inhibitors (Szmola, Kukor, & Sahin-Toth, 2003). Major three forms of digestive trypsins (anionic, meso and cationic trypsins) show a high sequence homology, Trypsin-3 differs only from Trypsin-1 at twenty-eight residues. The distinct conformation of Trypsin-3 is the result of evolutionary mutation in PRSS3 encoding Trypsin-3: the substitution of Gly¹⁹⁸ by Arg (Gly¹⁹³ for the trypsinogen form). Arg¹⁹³ is one of the most critical differences for Trypsin- 3 because it is located at the active site, at a position where almost all other serine protease members of the trypsin and chymotrypsin family possess a highly conserved glycine residue. Other unique Trypsin-3 residues, Lys⁷⁴ and Asp⁹⁷ are located on the periphery of the active site, and contribute to Trypsin-3 resistance to canonical trypsin inhibitors. These differences between Trypsin-3 and the other trypsins contribute to an unusually strong clustering of positive charges around the primary specificity pocket of Trypsin-3 (Katona, Berglund, Hajdu, Graf, & Szilagyi, 2002). These distinguishable structural particularities undoubtedly influence molecular partner binding and could reinforce potential inhibitory selectivity.

Although proteases show high similarity in the catalytic site, a high structural diversity could be conferred by loops surrounding the active site, resulting in slight but remarkably different three-dimensional arrangements (Goettig, Brandstetter, & Magdolen, 2019). Therefore, to achieve the identification of inhibitors with high selectivity, the binding surface interaction may not only cover the catalytic domain but also allosteric sites. Peptides are more likely to be able to bind to allosteric sites as well as to the active sites and thus be more selective compared to small molecules. The exquisite affinity and selectivity of antibodies in principle fulfils the binding properties that may account to distinguish closely related protease family members. Furthermore, the antibody approach offers a unique promise of being able to probe regions of the protease that are not restricted to the natural inhibitor binding site (that is, the active-site region) and thereby may facilitate the generation of allosteric inhibitors. Based on that we developed a strategy to identify single domain antibodies (SdAbs) able to selectively and efficiently inhibit human Trypsin-3. Single domain antibodies constitute interesting tools which are known to be highly resistant to degradation by proteases (Asaadi, Jouneghani, Janani, & Rahbarizadeh, 2021), easy to tailor and expressed in bacteria. More interestingly, several single domain antibodies (SdAbs) have been reported to bind concave surface of the antigen such as the active site or loops of an enzyme (Muyldermans, 2021).

Here, inventors identified SdAbs against human Trypsin-3 using subtractive phage display strategies. Using the most promising SdAbs as building blocks, they engineered biparatopic single domain antibodies to identify a highly selective binder that tightly inhibits the target protease.

SUMMARY OF THE INVENTION:

The present invention provides for an isolated anti Trypsin-3 single domain antibody, wherein said antibody specifically binds to human Trypsin-3 protein.

In a particular embodiment the antibody of the invention has at least one or more of the following properties: (i) it does not bind to with pro-form of Trypsin-3 or with mature Trypsin-2 and Trypsin- 1 isoforms and/or;

(ii) it further exhibits inhibition capacity of active human Trypsin-3 (neutralizing antibody) and/or;

(iii) it binds to human active Trypsin-3 protein with a KD of 200 nM or below, 100 nM or below, 10 nM or below, 9 nM or below, 8 nM or below, 7 nM or below, 6 nM or below, 5 nM or below, 4 nM or below, 3 nM or below, 2 nM or below, 1 nM or below, 0.1 nM or below, 0.05nM or below, 0.1 nM or below 0.05nM or below.

The invention also relates a polypeptide comprising at least one single-domain antibody according to the invention.

The invention further relates to an anti-Trypsin-3 single domain antibody according to the invention, a polypeptide comprising at least one single-domain antibody according to the invention used for the treatment of gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis.

The invention further relates to an anti-Trypsin-3 single domain antibody according to the invention, a polypeptide comprising at least one single-domain antibody according to the invention used for the treatment of cancer, especially cancer associated with Trypsin-3.

In a preferred embodiment the cancer is a tumor associated with Trypsin-3 activity.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, inventors disclosed single domain antibody (sdAb) (also called VHHs or Single domain antibodies), targeting specifically Trypsin-3 and not the other serine protease of the same family (Trypsin- 1 and Trypsin-2). Inventors generated, and selected single domain antibodies against Trypsin-3 obtained by phage display selection using a highly diverse synthetic library of single domain antibodies (Moutel et al., 2016). Because they are selected in vitro against the active form of the enzyme, single domain antibodies developed by this scheme have the inherent advantage of recognizing 3-D epitopes and the topography of the enzyme active site. The present data reflected a strong conformational selectivity towards only one form of a protease, the matured active form of Trypsin-3 since no interaction was detected between NT3 and other members of trypsin-like family as well as the proform of targeted protease Moreover, using the most promising sdAbs as building blocks, inventors engineered biparatopic single domain antibodies to identify a highly selective binder that tightly inhibit the target protease. This biparatopic single domain antibody specifically inhibits Trypsin-3 activity in malignant growth of PC3 prostate cancer cells and in gut tissue sections from IBS patients, providing a new tool in anti-Trypsin-3 immunotherapies.

Antibodies according to the invention

The present invention provides for an anti-Trypsin-3 single domain antibody (sdAb), wherein said antibody specifically binds to human Trypsin-3 protein. In some embodiments, the present invention provides for an anti-Trypsin-3 single domain antibody (sdAb), wherein said antibody specifically binds to human Trypsin-3 protein and does not bind to pro-form of Trypsin-3 and/or to mature Trypsin-2 and/or Trypsin- 1 isoform.

The single domain antibodies of the present invention preferably exhibit one or more additional desired functional properties selected from the group consisting of:

(i) it does not bind to with pro-form of Trypsin-3 and/or with mature Trypsin-2 and/or Trypsin- 1 polypeptide isoforms and/or;

(ii) it further exhibits inhibition capacity of active human Trypsin 3 protein (neutralizing antibody) and/or;

(iii) it binds to human active Trypsin-3 protein with a KD of 200 nM or below, 100 nM or below, 10 nM or below, 9 nM or below, 8 nM or below, 7 nM or below, 6 nM or below, 5 nM or below, 4 nM or below, 3 nM or below, 2 nM or below, 1 nM or below, 0.1 nM or below, 0.05nM or below.

As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody is also called VHH or “single domain antibody” or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0368 684, (Ward, Gussow, Griffiths, Jones, & Winter, 1989), (Holt, Herring, Jespers, Woolven, & Tomlinson, 2003) and WO 06/030220, WO 06/003388. VHHs have a molecular weight of about one-tenth of human IgG molecule ones and have a physical diameter of only a few nanometers. One consequence of the small size is the ability of single domain antibodies (or VHHs) to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., single domain antibodies (or VHHs) are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus, yet another consequence of small size is that a single domain antibody (or VHH) can inhibit activity/interactions as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compactness of the fold result in VHHs being extremely thermostable, stable to extreme pH and to proteolytic digestion, and the absence of Fc fragment provides a low antigenic character. Another consequence is that VHHs readily move from the circulatory system into tissues, and have a higher probability to cross the blood-brain barrier and can treat disorders that affect nervous tissue. Single domain antibodies (or VHHs) can further facilitate drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein 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 for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH (variable heavy chain) domains given by the International ImMunoGeneTics information system amino acid numbering (http://imgt.org/).

The term “Trypsin-3” also known as “Mesotrypsin” or “TRY3” means Trypsin-3 (EC 3.4.21.4) which is a serine protease that in humans is encoded by the PRSS3 gene. In humans, three serine protease (PRSS) genes encode trypsinogens: PRSS1 encodes Trypsinogen- 1 (cationic trypsin), PRSS2 encodes Trypsinogen-2 (anionic trypsin) and PRSS3 encodes Trypsinogen-3, wherein at least two isoforms with overlapping mature peptide sequences, formerly designated as mesotrypsinogen and trypsinogen IV, have been functionally characterized. The mature protein of PRSS3 gene uses the nomenclature of Trypsin-3 protein, common to all transcripts of this gene. Trypsin-3 is expressed in the brain and pancreas and is resistant to common trypsin inhibitors. It is active on peptide linkages involving the carboxyl group of lysine or arginine. Four transcript variants encoding different isoforms have been described for this gene. The whole sequence of human PRSS3 gene (gene encoding TRYPSIN- 3) is referenced as Gene ID: 5646. The protein sequence of said human Trypsin-3, and its isoforms, may be found in NCBI database with the following access numbers:

Trypsin-3 Variant 1 mRNA: NM_007343, and protein id: NP_031369 (isoform 1), (Trypsinogen 4)

Trypsin-3 Variant 2 mRNA: NM_002771 and protein id: NP_002762 (isoform 2) (Trypsinogen 3; Mesotrypsinogen)

Trypsin-3 Variant 3 mRNA: NM_001197097, and protein id: NP_001184026 (isoform

3),

Trypsin-3 Variant 4 mRNA NM_001197098, and protein id: NP_001184027 (isoform

4) (Trypsinogen 5).

Inventors determined that PRSS3 variant 1 is the major transcript expressed in intestinal epithelial cells and in colonic tissue samples. Nevertheless, 5 different transcripts encode the same active form of Trypsin-3 protein (P35030|TRY3_HUMAN Trypsin-3, P35030- 2|TRY3_HUMAN Isoform 2 of Trypsin-3, P35030-3|TRY3_HUMAN Isoform 3 of Trypsin-3, P35030-4|TRY3_HUMAN Isoform 4 of Trypsin-3, P35030-5|TRY3_HUMAN Isoform 5 of Trypsin-3).

Example of Trypsin-3 human amino acid sequence (mature active form) is provided in SEQ ID NO:29.

As used herein, the term "Affinity" refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, the stronger the affinity. Affinity can be determined by measuring KD. As used herein, the term KD is intended to refer to the dissociation constant, which is obtained from the ratio of Koir to K_on (i.e. Koir / K_on) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the Art. One method for determining the KD of an antibody is by using surface Plasmon resonance, using a biosensor system such as a Biacore® system.

In a specific embodiment, the affinity of the antibody the invention with human Trypsin- 3 protein refers to an antibody that has a KD of 200 nM or below, 100 nM or below, preferably at least 10 nM or below, 9 nM or below, 8 nM or below, more preferably 7 nM or below, 6 nM or below, 5 nM or below, 4 nM or below, 3 nM or below, 2 nM or below, at least 1 nM or below, and even more preferably 0.1 nM or less, 0.05 nM or less (for bispecific antibodies) as measured in “Affinity measurement” using SPR (Surface Plasmon Resonance) technology as described in more detail in the Examples below.

In some embodiments, the single domain antibody is a “humanized” single domain antibody.

As used herein the term “humanized” refers to a single domain antibody of the invention wherein an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a variable heavy chain (VH) from a conventional chain antibody from a human being. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized single domain antibodies still retain the favourable properties of single domain antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions. For example, the single domain antibodies of the invention may be suitably humanized at any framework residue depicted in Table 7 provided that the single domain antibodies remain soluble and do not significantly lose their affinity for Trypsin-3.

Single domain antibody (NT3-X) and derivative

In particular, the inventors developed 5 fully human single domain antibodies (called also here NT3-1, NT3-3, NT3-7, NT3-12 and NT3-16) against human Trypsin 3 said single domain antibodies was selected to target very specifically human active Trypsin-3 with high affinity (KD~ 10 nM or below, ~7 nM or below, ~ 5 nM or below, ~3 nM or below, ~2 nM or below, and not the other serine protease of the same family (Trypsin- 1 and Trypsin-2) or not the pro-form of Trypsin-3 polypeptide. Furthermore, inventors characterized some of them as specific Trypsin-3 protease inhibitor (NT3-7, NT3-12).

Thus, in some embodiment, the single-domain antibody of the present invention is a Trypsin-3 neutralizing single-domain antibody. Which inhibit the protease activity of Trypsin- 3 (see below “The biological activities of the antibody of the invention using Protease activity assay”).

As used herein, "neutralizing antibody" refers to an antibody, for example, a single domain antibody, capable of binding and inhibiting the active form of Trypsin-3 protein and such as the correction of intestinal permeability and pain observed in gut diseases associated with such impairment such as, in Irritable Bowel Syndrome (IBS) including gluten hypersensitivity. In a specific embodiment, a neutralizing antibody refers to an antibody that has an IC50 of at least 100 pM or below, preferably at least 50 pM or below, more preferably at least 10 pM or below, even more preferably at least 1 pM or below as measured in “ Characterization of Trypsin-3 Single domain antibodies” as protease inhibitor” using Protease Activity assay as described in more detail in the Examples below.

In some embodiments the isolated anti-Trypsin-3 single domain antibody according to the invention wherein said single domain antibody comprise

(a) a CDR1 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 2, a CDR2 of NT3- 7 sdAb having a sequence set forth as SEQ ID NO: 3, and a CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; or

(b) a CDR1 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 6, a CDR2 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 7, and a CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 8; or

(c) a CDR1 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 10, a CDR2 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 11, and a CDR3 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 12; or

(d) comprise a CDR1 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 14, a CDR2 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 15, and a CDR3 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 16; or

(e) a CDR1 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 18, a CDR2 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 19, and a CDR3 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 20.

In a particular embodiment, the invention relates to an anti-Trypsin-3 single domain antibody wherein the single domain antibody comprising: a variable heavy chain (VH) of having at least 70% of identity with sequence set forth as SEQ ID NO: 1 (NT3-7) or a variable heavy chain (VH) having at least 70% of identity with sequence set forth as SEQ ID NO:5 (NT3-12) or a variable heavy chain (VH) of having at least 70% of identity with sequence set forth as SEQ ID NO:9 (NT3-1) or a variable heavy chain (VH) having at least 70% of identity with sequence set forth as SEQ ID NO: 13 (NT3-3) a variable heavy chain (VH) having at least 70% of identity with sequence set forth as SEQ ID NO: 17 (NT3-16)

According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% of identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as CLUSTAL or BLAST P (Karlin & Altschul, 1990).

In a particular embodiment, the isolated single domain antibody according to the invention has the sequence of variable heavy chain (VH) set forth as SEQ ID NO: l(“NT3-7”); as SEQ ID NO:5 (“NT3-12”) as SEQ ID NO:9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”).

In a particular embodiment, the single domain antibodies described above binds to the same antigen and have the same or improved properties (see specific Trypsin-3 binder and/or specific protease inhibitor activity) of the single domain antibody of the invention i.e. the antibody with the CDRs of SEQ ID NO: 2 to 4; (“NT3-7”); the antibody with the CDRs of SEQ ID NO: 6 to 8; (“NT3-12”); the antibody with the CDRs of SEQ ID NO: 10 to 12; (“NT3-1”); the antibody with the CDRs of SEQ ID NO: 14 to 16; (“NT3-3”); the antibody with the CDRs of SEQ ID NO: 18 to 20; (“NT3-16”).

The sequences NT3-7 are described below in Table 1 for the variable heavy chain (VH) and CDRs domains (or FRs) of Single Domain antibody NT3-7.

TABLE 1

The sequences of NT3-12 are described below in Table 2 for the variable heavy chain (VH) and CDRs domains of Single Domain antibody NT3-12.

TABLE 2

The sequences NT3-1 are described below in Table 3 for the variable heavy chain (VH) and CDRs domains (or FRs) of Single Domain antibody NT3-1.

TABLE 3

The sequences NT3-3 are described below in Table 4 for the variable heavy chain (VH) and CDRs domains (or FRs) of Single Domain antibody NT3-3. TABLE 4

The sequences NT3-16 are described below in Table 5 for the variable heavy chain (VH) and CDRs domains (or FRs) of Single Domain antibody NT3-16.

TABLE S

• F uncti onal vari ant NT3 -X The present invention thus provides antibodies comprising functional variants of the VH region including FRs and/or one or more CDRs of single domain antibody of the invention. A functional variant of a VH (FR, or CDR) used in the context of a single domain antibody of the present invention still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody (i.e. single domain antibody (sdAb) NT3-X NT3- X selected from NT3-1, NT3-3, NT3-7, NT3-12 and NT3-16) and in some cases such a single domain antibody of the present invention may be associated with greater affinity, selectivity and/or specificity than the parent single domain antibody (or VHH). Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs ((Yang et al., 1995), chain shuffling (Marks et al., 1992), use of mutator strains of E. coli (Low, Holliger, & Winter, 1996), DNA shuffling (Patten, Howard, & Stemmer, 1997), phage display (Thompson et al., 1996) and sexual PCR (Crameri, Raillard, Bermudez, & Stemmer, 1998). Such functional variants typically retain significant sequence identity to the parent single domain antibody (or VHH). The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements. The sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance, at least 3, such as at least, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:

Aliphatic residues I, L, V, and M

Cycloalkenyl-associated residues F, H, W, and Y

Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E

Polar residues C, D, E, H, K, N, Q, R, S, and T

Positively charged residues H, K, and R

Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S

Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T

Flexible residues Q, T, K, S, G, P, D, E, and R

More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of single domain antibody of the invention. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 1 and Gap extension= 1). Suitable variants typically exhibit at least about 70% of identity to the parent protein. According to the present invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 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% of identity with the second amino acid sequence. According to the present invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.

In some embodiments, the single domain antibody of the present invention is a single domain antibody having a variable heavy chain comprising i) a VH-CDR1 having at least 3, 2, 1 conservative substitutions within the VH-CDR1 of single domain antibody NT3-X (SEQ ID N°2 or SEQ ID N°6 or SEQ ID N°10 or SEQ ID N°14 or SEQ ID N°18), ii) a VH-CDR2 having at least having at least 3, 2, 1 conservative substitutions within the VH-CDR2 of single domain antibody NT3-X (SEQ ID N°3 or SEQ ID N°7 or SEQ ID N°11 or SEQ ID N°15 or SEQ ID N°19) and iii) a VH-CDR3 having at least, 3, 2, 1 conservative substitutions within the VH- CDR3 of single domain antibody NT3-X (SEQ ID N°4 or SEQ ID N°8 or SEQ ID N°12 or SEQ ID N°16 or SEQ ID N°20).

As used herein, a “NT3 analogue” or “NT3 derivative” refers to a single domain antibody exhibiting at least the same, or better, specific binding to Trypsin-3 protein and at least one of the biological activities of a single domain antibody NT3 with a VH as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO: 5 (“NT3-12”) as SEQ ID NO: 9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”). The NT3 analogue may for example be characterized in that it is capable of inhibiting capacity of active Trypsin-3 protein through experiments (see Example 1 : Material and Method /Protease Activity ). Briefly, the Trypsin-3 activity assay may be performed using Human active Trypsin-3, by incubating the NT3 analogue. A Trypsin-3 substrate (such as N-p-Tosyl-GPR-amino-4-methylcoumarin hydrochloride) is added after incubation and substrate degradation is calculated by the change in fluorescence on a microplate reader.

The biological activities of the antibody of the invention are, for example, to reduce the level of Trypsin-3 proteolytic activity as described above. The evaluation of the Trypsin-3 activity level allows to determine the therapeutic properties of the single domain antibody (neutralizing antibody) of the invention such as the correction of intestinal permeability and pain observed in gut diseases associated with such impairment such as, in Irritable Bowel Syndrome (IBS) including gluten hypersensitivity.

Said antibodies may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York). For example, the BIACORE® (GE Healthcare, Piscataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.

Single-domain Antibody which compete with the single-domain antibody of the invention

A further aspect of the invention refers to a cross-competing single-domain antibody which cross-competes for binding Trypsin-3 with the single-domain antibody of the invention. In some embodiment, the cross-competing single-domain antibody of the present invention cross-competes for binding Trypsin-3 with the single-domain antibody comprising: (a) a CDR1 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 2, a CDR2 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 3, and a CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; or

(d) a CDR1 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 14, a CDR2 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 15, and a CDR3 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 16; or

In some embodiment, the cross-competing single-domain antibody of the present invention cross-competes for binding Trypsin-3 with the single-domain antibody comprising or consisting a variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO:5 (“NT3-12”) as SEQ ID NO: 9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”).

As used herein, the term “cross-competes” refers to single-domain antibodies which share the ability to bind to a specific region of an antigen. In the present disclosure the singledomain antibody that “cross-competes" has the ability to interfere with the binding of another single-domain antibody for the antigen in a standard competitive binding assay. Such a singledomain antibody may, according to non-limiting theory, bind to the same or a related or nearby (e.g., a structurally similar or spatially proximal) epitope as the single-domain antibody with which it competes. Cross-competition is present if single-domain antibody A reduces binding of single-domain antibody B at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said single-domain antibodies. As the skilled artisan appreciates competition may be assessed in different assay set-ups. One suitable assay involves the use of the Biacore technology (e.g., by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore, a high throughput process for "binning" antibodies based upon their cross-competition is described in International Patent Application No. WO2003/48731.

According to the present invention, the cross-competing antibody as above described retain the activity of the single-domain antibody which comprises

(a) a CDR1 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 2, a CDR2 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 3, and a CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; or

According to the present invention, the cross-competing antibody as above described retain the activity of the single-domain antibody comprising or consisting a variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO:5 (“NT3-12”) as SEQ ID NO:9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”).

Thus, in some embodiment, the cross-competing single-domain antibody of the present invention is a Trypsin-3 neutralizing single-domain antibody which inhibit the protease activity of Trypsin-3.

In some embodiments, the cross-competing single-domain antibody of the present invention bound to either recombinant or human Trypsin-3 protein and does not bind to with pro-form of Trypsin-3 or with mature Trypsin-2 and Trypsin-1 isoforms.

Single domain antibody polypeptide and derivatives

• monospecific

A further aspect of the invention refers to a polypeptide comprising at least one single domain antibody of the invention. Typically, the polypeptide of the invention comprises a single domain antibody of the invention, which is fused at its N terminal end, at its C terminal end, or both at its N terminal end and at its C terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein. According to the invention the polypeptides that comprise a sole single domain antibody are referred to herein as "monovalent" polypeptides. Polypeptides that comprise or essentially consist of two or more single domain antibodies according to the invention are referred to herein as "multivalent" polypeptides.

In some embodiments, the two or more single domain antibodies according to the invention (“multivalent" polypeptides) can be linked to each other directly (i.e. without use of a linker) or via a linker.

The linker is typically a linker peptide and will, according to the invention, be selected so as to allow binding of the two single domain antibodies to the same epitopes of two different Trypsin-3 proteins. Suitable linkers inter alia depend on the epitopes and, specifically, the distance between the epitopes on two different Trypsin-3 proteins to which the single domain antibodies bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation. Also, when the two single domain antibodies that bind to two different Trypsin-3 proteins may also be linked to each other via a third single domain antibody (in which the two single domain antibodies may be linked directly to the third domain antibody or via suitable linkers). Such a third single domain antibody may for example be a single domain antibody that provides for an increased half-life. For example, the latter single domain antibody may be a single domain antibody that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more single domain antibodies that bind to different Trypsin-3 protein are linked in series (either directly or via a suitable linker) and the third (single) single domain antibody (which may provide for increased half-life, as described above) is connected directly or via a linker to one of these two or more aforementioned single domain antibodies.

Suitable linkers are described herein in connection with specific polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides. The linker sequence may be a naturally occurring sequence or a non- naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non- immunogenic in the subject to which the anti- Trypsin-3 protein polypeptide of the invention is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly/Ser linkers of different length including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

According to a specific embodiment, the at least two single domain antibodies according to the invention (“monospecific multivalent" polypeptides) are connected with linkers derived from the hinge region of heavy chain antibodies. Such polypeptides are also called “minibody” The term "minibody" corresponds to an antibody format containing the CH3 domain of the Fc fragment (from classic Ig) followed by a hinge sequence fused to a VHH (or a ScFv domain (example of “minibody” with ScFv domain are described in (Hu et al., 1996; Kim et al., 2014; Nunez-Prado et al., 2015) and WO 94/04678.

According to the invention, the single domain antibodies and polypeptides of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The single domain antibodies and polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The single domain antibodies and polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433 A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

• Multispecific

In some embodiments, the polypeptide comprises at least one single domain antibody of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is typically also a single domain antibody. Such a polypeptide is referred to herein as "multi specific" polypeptide; in opposition to a polypeptide comprising the same single domain antibodies (“monospecific” polypeptide). Thus, in some embodiments, the polypeptide of the invention may also provide at least one further binding site directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope. Said binding site is directed against to the same protein, polypeptide, antigen, antigenic determinant or epitope for which the single domain antibody of the invention is directed against, or may be directed against a different protein, polypeptide, antigen, antigenic determinant or epitope) from the single domain antibody of the invention.

Typically, the one or more further binding site may comprise one or more parts, fragments or domains of conventional chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, a single domain antibody of the invention may be linked to a conventional (typically human) VH or VL optionally via a linker sequence.

A "bispecific" polypeptide of the invention is a polypeptide that comprises at least one single domain antibody directed against a first antigen (i.e. Trypsin-3 protein) and at least one further binding site directed against a second antigen (i.e. different from Trypsin-3 protein), whereas a "trispecific" polypeptide of the invention is a polypeptide that comprises at least one single domain antibody directed against a first antigen (i.e. Trypsin-3 protein), at least one further binding site directed against a second antigen (i.e. different from Trypsin-3 protein) and at least one further binding site directed against a third antigen (i.e. different from both i.e. first and second antigen); etc.

In some embodiments, the polypeptide is as described in W02006064136. In particular the polypeptide may consist of i) a first fusion protein wherein the CL constant domain of an antibody is fused by its N-terminal end to the C-terminal end to a single domain antibody according to the invention (i.e. a single antibody directed against Trypsin-3 protein) and ii) a second fusion protein wherein the CHI constant domain of an antibody is fused by its N- terminal end to the C-terminal end of a single domain antibody directed against an antigen different from Trypsin-3 protein. In another particular embodiment, the polypeptide consists of a first fusion protein wherein the CHI constant domain of an antibody is fused by its N-terminal end to the C-terminal end of a single domain antibody directed against an antigen different from Trypsin-3 protein and a second fusion protein wherein the CL constant domain of an antibody is fused by its N-terminal end to the C-terminal end to a single domain antibody of the invention (i.e. Trypsin-3 protein). In some embodiments, the polypeptide is a biparatopic polypeptide. As used herein, the term "biparatopic" polypeptide means a polypeptide comprising a single domain antibody and a second single domain antibody as herein defined, wherein these two single domain antibodies are capable of binding to two different epitopes of one antigen (e.g. Trypsin-3 protein), which epitopes are not normally bound at the same time by one monospecific immunoglobulin, such as e.g. a conventional antibody or one single domain antibody. The biparatopic polypeptides according to the invention are composed of single domain antibodies which have different epitope specificities, and do not contain mutually complementary variable domain pairs which bind to the same epitope. They do therefore not compete with each other for binding to Trypsin- 3 protein.

In some embodiments, the two single domain antibodies of the biparatopic polypeptide of the present invention can be linked to each other directly (i.e. without use of a linker) or via a linker.

The linker is typically a linker peptide and will, according to the invention, be selected so as to allow binding of the two single domain antibodies to each of their at least two different epitopes of Trypsin-3 protein. Suitable linkers inter alia depend on the epitopes and, specifically, the distance between the epitopes on Trypsin-3 protein to which the single domain antibodies bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation. Also, when the two single domain antibodies that bind to Trypsin-3 protein may also be linked to each other via a third single domain antibody (in which the two single domain antibodies may be linked directly to the third domain antibody or via suitable linkers). Such a third single domain antibody may for example be a single domain antibody that provides for an increased half-life. For example, the latter single domain antibody may be a single domain antibody that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more single domain antibodies that bind to Trypsin-3 protein are linked in series (either directly or via a suitable linker) and the third (single) single domain antibody (which may provide for increased half-life, as described above) is connected directly or via a linker to one of these two or more aforementioned single domain antibodies. Suitable linkers are described herein in connection with specific polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non- immunogenic in the subject to which the anti- Trypsin-3 protein polypeptide of the invention is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly/Ser linkers of different length including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

In some embodiments, the polypeptide comprises at least one single-domain antibody according to the present invention.

In some embodiments, the polypeptide comprises at least two single-domain antibodies according to the present invention.

In some embodiments, the polypeptide comprises two single-domain antibodies according to the invention.

In some embodiments, the polypeptide of the present invention comprises at least one single-domain antibody comprising

In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies comprising (a) a CDR1 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 2, a CDR2 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 3, and a CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; and/or

(b) a CDR1 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 6, a CDR2 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 7, and a CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 8; and/or

(c) a CDR1 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 10, a CDR2 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 11, and a CDR3 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 12; and/or

(d) a CDR1 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 14, a CDR2 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 15, and a CDR3 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 16; and/or

In some embodiments, the polypeptide of the present invention comprises 2, 3, 4 or 5 single-domain antibodies comprising

(a) a CDR1 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 2, a CDR2 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 3, and a CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; and/or

(e) a CDR1 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 18, a CDR2 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 19, and a CDR3 of NT3-16 sdAb having a sequence set forth as SEQ ID NO: 20. In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% of identity with sequence set forth as SEQ ID NO:4.

In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% of identity with the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO:5 (“NT3-12”) as SEQ ID NO:9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”).

In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); and/or as SEQ ID NO:5 (“NT3-12”) and/or as SEQ ID NO:9 (“NT3-1”) and/or as SEQ ID NO: 13 (“NT3-3”); and/or as SEQ ID NO: 17 (“NT3-16”).

In some embodiments, the polypeptide of the present invention comprises 2, 3, 4 or 5 single-domain antibodies having the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); and/or as SEQ ID NO:5 (“NT3-12”) and/or as SEQ ID NO:9 (“NT3-1”) and/or as SEQ ID NO: 13 (“NT3-3”); and/or as SEQ ID NO: 17 (“NT3-16”).

According to the present invention, the polypeptide of the present invention as above described retain the biological activity of the single-domain antibody comprising or consisting a variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO:5 (“NT3- 12”) as SEQ ID N0:9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3- 16”).

Thus, in some embodiment, the polypeptide of the present invention comprises at least one Trypsin-3 neutralizing single-domain antibody (which inhibit the protease activity of Trypsin-3) such as NT3-7 or NT3-12.

In some embodiments, the polypeptide of the present invention comprises a sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”) and/or a sequence of variable heavy chain (VH) set forth as SEQ ID NO: 5 (“NT3-12”).

In some embodiments, the polypeptide of the present invention comprises

(b) a CDR1 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 6, a CDR2 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 7, and a CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 8. In some embodiments, the polypeptide of the present invention bound to either recombinant or human Trypsin-3 protein and does not bind to with pro-form of Trypsin-3 or with mature Trypsin-2 and Trypsin- 1 isoforms.

In specific embodiment with biparatopic Trypsin-3 single domain antibodies (with any single domain antibodies of the invention and especially with “NT3-7” and “NT3-12” single domain antibodies) and as demonstrate in experimental, an in silico analysis based on Trypsin- 3 model of the 3D-structure was performed to optimize the linker favoring the interaction between both single domain antibodies on Trypsin-3 target. A linker of a potential length of 70.5 A have been designed to allow flexibility of the bioactive biparatopic molecule.

Thus, in this specific embodiment, the linker between single domain antibodies “NT3-7” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1) and “NT3-12” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 5) single domain antibodies, has length of 70.5 A.± 5.

The inventors furthermore designed the sequence of the linker to avoid amino acids known as predicted protease sensitive-site.

In a specific embodiment the linker between single domain antibodies of the invention especially with “NT3-7” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1) and “NT3-12” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 5) single domain antibodies, has the following sequence:

GGGGSGGGGSAGSAAGSGEGGGGSGGGGSGGG (SEQ ID N° 21).

In another specific embodiment the linker between single domain antibodies of the invention especially with “NT3-7” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1) and “NT3-12” (sequence of variable heavy chain (VH) set forth as SEQ ID NO: 5) single domain antibodies, has the following sequence: GGGSGGGSAGSAAGSGEGGGSGGG (SEQ ID NO: 30).

Methods of producing antibodies of the invention and immunoconjugates

Methods for obtaining such antibodies are well known in the art.

Camel Ig can be modified by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody -derived protein known as a "single domain antibody" or “VHH”. See U.S. patent number 5,759,808 issued June 2, 1998; see also (Cortez-Retamozo et al., 2002; Dumoulin et al., 2003; Lauwereys et al., 1998; Pleschberger et al., 2003; Stijlemans et al., 2004). Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium / Sanofi, Gentilly, France. In certain embodiments herein, the single-chain camelid antibody or single domain antibody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with Trypsin-3 protein or a peptide fragment thereof, using techniques described herein for other antibodies. The Trypsin-3 protein-binding camelid single domain antibody (VHH) is next engineered from the camelid single-chain antibodies. Selection is performed for example from a library of phage displaying appropriately mutagenized camelid single domain antibody (VHH) proteins using panning procedures with Trypsin-3 protein as a target. Alternatively, VHHs can be selected from a naive phage library (without immunization) using panning procedures with Trypsin-3 protein as a target (see (Moutel et al., 2016)).

A single domain antibody of the invention can be conjugated with a detectable label to form an immunoconjugate. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are 3H, 1251, 13 II, 35S and 14C.

Immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently -labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, immunoconjugates can be detectably labeled by coupling an antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.

Alternatively, immunoconjugates can be detectably labeled by linking an antibody to an enzyme. When the enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include P- galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.

An antibody of the invention may be labelled with a metallic chemical element such as lanthanides. Lanthanides offer several advantages over other labels in that they are stable isotopes, there are a large number of them available, up to 100 or more distinct labels, they are relatively stable, and they are highly detectable and easily resolved between detection channels when detected using mass spectrometry. Lanthanide labels also offer a wide dynamic range of detection. Lanthanides exhibit high sensitivity, are insensitive to light and time, and are therefore very flexible and robust and can be utilized in numerous different settings. Lanthanides are a series of fifteen metallic chemical elements with atomic numbers 57-71. They are also referred to as rare earth elements. Lanthanides may be detected using CyTOF technology. CyTOF is inductively coupled plasma time-of-flight mass spectrometry (ICP-MS). CyTOF instruments are capable of analyzing up to 1000 cells per second for as many parameters as there are available stable isotope tags.

Those of skill in the art will know of other suitable labels which can be employed in accordance with the present invention. The binding of marker moieties to single domain antibodies can be accomplished using standard techniques known to the art.

Moreover, the convenience and versatility of immunochemical detection can be enhanced by using monoclonal antibodies that have been conjugated with avidin, streptavidin, and biotin.

The single domain antibodies of the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’ s instructions. Alternatively, antibodies of the invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.

Accordingly, a further object of the invention relates to a nucleic acid sequence encoding a single domain antibody according to the invention.

In a particular embodiment, the invention relates to a nucleic acid sequence encoding the VH domain of the antibody of the invention (e.g. single domain antibody NT3-X). Example of nucleic sequence encoding the VH of NT3 in a plasmid are described in SEQ ID NO:24 (NT3-1), in SEQ ID NO:25 (NT3-3), in SEQ ID NO:26 (NT3-7), in SEQ ID NO:27 (NT3-12), in SEQ ID NO:28 (NT3-16).

Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

So, a further object of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like. Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.

A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention and expressing a single domain antibody according to the invention. Accordingly, such recombinant host cells can be used for the production of antibodies of the invention.

The term “transformation” means the introduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been “transformed”.

The nucleic acids of “the invention” may be used to produce an antibody of the invention in a suitable expression system. The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli. Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) is defective (Urlaub & Chasin, 1980), rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as “YB2/0 cell”), and the like.

The present invention also relates to a method of producing a single domain antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained (iii), recovering the expressed antibody.

Detecting and Diagnostic methods of the invention:

Single Domain antibodies of the present invention and immunoconjugates can be used for detecting human Trypsin-3 protein, and/or evaluating its amount in a biological sample, in particular a culture medium sample, a whole blood sample, a serum sample, a plasma sample, , or any tissue sample. Therefore, they can be used for diagnosing all diseases associated with abnormal Trypsin-3 activity. Accordingly, the method of detection of the invention is consequently useful for the in vitro diagnosis of Trypsin-3 associated pathologies.

The term “Trypsin-3 associated pathologies” has its general meaning in the art and refers to a disease characterized by abnormal Trypsin-3 activity. Trypsin-3 associated pathologies include among others, gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis and also cancer especially cancer associated with Trypsin-3.

The terms "cancer" and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the use of the invention, diseases, namely tumors that express/secrete Trypsin-3 are most likely to be detected or respond the Trypsin-3 single domain antibodies of the present invention. In particular, the cancer may be associated with a solid tumor or lymphoma/leukemia (from hematopoietic cell). Examples of cancers that are associated with solid tumor formation include breast cancer, uterine/cervical cancer, oesophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, nonsmall cell lung cancer, stomach cancer, tumors of mesenchymal origin (i.e; fibrosarcoma and rhabdomyoscarcoma) tumors of the central and peripheral nervous system (i.e; including astrocytoma, neuroblastoma, glioma, glioblatoma) and/or thyroid cancer.

In a particular embodiment the solid tumor is a cancer associated with Trypsin-3.

The term “cancer associated with Trypsin-3” refers namely tumors that express secrete Trypsin-3. Examples of such cancer associated with Trypsin-3 include prostate cancer (Hockla A. Mol Cancer Res. 2012 December ; 10(12): 1555-1566. Doi: 10.1158/1541-7786.MCR-12- 0314.), colon adenocarcinoma (Zhang, Appl Immunohistochem Mol Morpho 2021, Mar 22), lung adenocarcinoma (MA, scientific reports (2019) 9: 1844), pancreatic cancer (Jiang G. Gut 2010;59:1535el544. Doi: 10.1136/gut.2009.200105), ovarian cancer (MA R., Gynecologic Oncology 137 (2015) 546-552), breast cancer (Qian L., Oncotarget, 2017, Vol. 8, (No. 13), pp: 21444-21453), Gastric cancer (Fei Wang MS, J Surg Oncol. 2019;119: 1108-1121.), endometrial cancer (Aboulouard Cell Reports Medicine 2, 100318 June 15, 2021 a 2021), large B cell lymphoma (Hindawi Disease Markers Volume 2022, Article ID 1254790, 9 pages), oesophagal cancer (Han S, Lee CW, Trevino JG, Hughes SJ, Sarosi GA Jr (2013), PloS ONE 8(10): e76667. Doi:10.1371/journal.pone.0076667).

Preferably the solid tumor and cancer associated with Trypsin-3 is selected from the group consisting of pancreatic cancer and prostate cancer.

More preferably the pancreatic cancer is pancreatic ductal adenocarcinoma. An object of the invention is a method for detecting human Trypsin-3 protein and/or evaluating their amount in a biological sample, wherein said method comprises contacting said sample with an antibody or immunoconjugate of the invention under conditions allowing the formation of an immune complex between human Trypsin-3 protein and said antibody/immunoconjugate, and detecting or measuring the immune complex formed.

The immune complex formed can be detected or measured by a variety of methods using standard techniques, including, by way of non-limitative examples, enzyme-linked immunosorbent assay (ELISA) or other solid phase immunoassays, radioimmunoassay, electrophoresis, immunofluorescence, or Western blot.

A further object of the invention is a method for diagnosing a Trypsin-3 associated pathologies, wherein said method comprising evaluating the amount of human Trypsin-3 protein, as indicated above, in a biological sample from a subject to be tested, and comparing the determined amount with a control value of Trypsin-3 in a normal subject.

Finally, the invention also provides kits comprising at least one single domain antibody of the invention. Kits of the invention can contain a single domain antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., Sepharose beads). Kits can be provided which contain antibodies for detection and quantification of Trypsin-3 protein in vitro, e.g. in an ELISA or a Western blot. Such single domain antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.

Therapeutic methods of the invention:

Any of the anti-Trypsin-3, in particular neutralizing, single domain antibody; of the cross-competing anti-Trypsin-3, in particular neutralizing, single-domain antibody; of the polypeptide comprising at least one anti-Trypsin-3, in particular neutralizing, single-domain antibody; of the nucleic acid sequence encoding an anti-Trypsin-3, in particular neutralizing, single domain antibody; or of the vector comprising a nucleic acid encoding an anti-Trypsin-3, in particular neutralizing, single domain antibody previously described may be use for therapeutic purposes according to the invention. A further object of the invention relates to a pharmaceutical composition comprising an anti-Trypsin-3 neutralizing single domain antibody of the invention, or the cross-competing neutralizing single-domain antibody of the invention, or the polypeptide comprising at least one neutralizing single-domain antibody of the invention or a nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention or a vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention. Thus, in some embodiment, the pharmaceutical composition of the present invention comprises at least one Trypsin-3 neutralizing single-domain antibody (which inhibit the protease activity of Trypsin-3) such as NT3-7 or NT3-12 or the cross-competing neutralizing single-domain antibody of the invention , or the polypeptide comprising at least one neutralizing single-domain antibody of the invention such as NT3-7 or NT3-12 or a nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention such as NT3- 7 or NT3-12 or a vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention such as NT3-7 or NT3-12.

A further object of the invention relates to a pharmaceutical composition comprising an anti-Trypsin-3 neutralizing single domain antibody of the invention or the cross-competing anti-Trypsin-3 neutralizing single-domain antibody of the invention, or the polypeptide comprising at least one anti-Trypsin-3 neutralizing single-domain antibody of the invention or a nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention or a vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention for use in therapy.

A further object of the invention relates to a pharmaceutical composition comprising an anti-Trypsin-3 neutralizing single domain antibody of the invention the cross-competing anti- Trypsin-3 neutralizing single-domain antibody of the invention, or the polypeptide comprising at least one anti-Trypsin-3 neutralizing single-domain antibody of the invention or a nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention or a vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention for use in the treatment of Trypsin-3 associated pathologies.

As previously described, the term "Trypsin-3 associated pathologies" has its general meaning in the art and refers to a disease characterized by abnormal Trypsin-3 activity. Trypsin- 3 associated pathologies include among others, gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis and also cancer, especially cancer associated with Trypsin-3.

A further object of the invention relates to a method for treating a Trypsin-3 associated pathology comprising administering a subject in need thereof with a therapeutically effective amount of an anti-Trypsin-3 neutralizing single domain antibody of the invention, the crosscompeting anti-Trypsin-3 neutralizing single-domain antibody of the invention, or the polypeptide comprising at least one anti-Trypsin-3 neutralizing single-domain antibody of the invention or the nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention or the vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of the invention.

In some embodiments, the present invention relates to a method of treating a subject suffering from a trypsin-3 associated pathology by administering an anti-trypsin-3 neutralizing single domain antibody, a cross-competing anti-Trypsin-3 neutralizing single-domain antibody or a polypeptide comprising at least one (i.e. 1, 2, 3, 4, 5 or more) anti-Trypsin-3 neutralizing single-domain antibody, said neutralizing single-domain antibody comprising means for binding human trypsin-3, wherein said anti -trypsin-3 neutralizing single domain antibody :

(i) does not bind to pro-form of Trypsin-3 and/or to mature Trypsin-2 and/or Trypsin- 1 isoform

(ii) further exhibits inhibition capacity of active human Trypsin-3 (neutralizing antibody); and/or

(iii) binds to human active Trypsin-3 protein with a KD of 200 nM or below, 100 nM or below, 10 nM or below, 9 nM or below, 8 nM or below, 7 nM or below, 6 nM or below, 5 nM or below, 4 nM or below, 3 nM or below, 2 nM or below, 1 nM or below, 0.1 nM below, 0.05nM or less below.

In some embodiments, the anti-Trypsin-3 neutralizing single domain antibody, the cross-competing anti-Trypsin-3 neutralizing single-domain antibody or the polypeptide comprising at least one anti-Trypsin-3 neutralizing single-domain antibody comprises:

(b) a CDR1 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 6, a CDR2 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 7, and a CDR3 of NT3- 12 sdAb having a sequence set forth as SEQ ID NO: 8.

In some embodiments, the polypeptide comprises at least one single-domain antibodies having at least 70% of identity with the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); and/or as SEQ ID NO: 5 (“NT3-12”).

In some embodiments, the polypeptide of the present invention comprises at least two singledomain antibodies having the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); and/or as SEQ ID NO:5 (“NT3-12”).

By a "therapeutically effective amount" of the single domain antibody of the invention is meant a sufficient amount of the single domain antibody to treat said Trypsin-3 associated pathology, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

For administration, the single domain antibody of the invention or the fragment thereof is formulated as a pharmaceutical composition. A pharmaceutical composition comprising an antibody of the invention or a fragment thereof can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular. To prepare pharmaceutical compositions, an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The pharmaceutical forms include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. A single domain antibody of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

The single domain antibodies of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

Use of a food-grade bacterium

Use of a food-grade bacterium (or probiotic bacterium) to deliver an anti-Trypsin-3 single domain antibody of the present invention or polypeptide comprising at least one anti- Trypsin-3 single antibody and especially an anti- Trypsin-3 neutralizing single domain antibody of the invention or polypeptide comprising at least one anti-Trypsin-3 neutralising single domain antibody of the invention allow to provides a safety and a good efficiency to deliver these compound locally in the gut for diagnostic or therapeutic purpose.

Accordingly further object of the invention relates to a recombinant food-grade bacterium comprising a gene or nucleic sequence coding for a an anti- Trypsin-3 single domain antibody of the present invention or polypeptide comprising at least one anti- Trypsin-3 single antibody of the invention (such as NT3-X sdAB) for use 1) to detect and/or diagnosis “Trypsin- 3 associated pathologies” (such as gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis) and/or 2) in the treatment of a patient affected with gut diseases associated with intestinal permeability (IBD, IBS, celiac disease or pouchitis), said recombinant food-grade bacterium being administered locally in the gut of the patient to be treated. Accordingly, in order to be administered directly to the intestine (gut), the recombinant food-grade bacterium according to the invention is administered to the IBD or IBS patient preferably orally (including buccal and sublingual administration), rectally or topically (intracolic administration),

Another aspect of the invention relates to a therapeutic composition comprising a recombinant food-grade bacterium as defined above.

Another aspect of the invention relates to a an anti- Trypsin-3 neutralizing single domain antibody of the invention or a polypeptide comprising at least one anti-Trypsin-3 neutralizing single domain antibody of the invention for use in the treatment of a patient affected with gut diseases associated with intestinal permeability such as, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis wherein a food-grade bacterium comprising a gene or nucleic sequence coding for an anti-Trypsin-3 single domain antibody of the present invention or polypeptide comprising at least one anti-Trypsin-3 neutralizing single antibody (such as NT3-7 and/or NT3-12 sdAB) is used to deliver directly the neutralizing sdAb or polypeptide comprising at least one anti-Trypsin-3 neutralizing in the gut.

As used herein, the term “food-grade bacterium” denotes a bacterium that is widely used in fermented foods and possesses a perfect safety profile recognized by the GRAS (Generally Recognized As Safe) and QPS (Qualified Presumption of Safety) status in USA and European Community, respectively. Such bacterium can be safely in functional foods or food additives with allegations concerning maintain in good health and well-being or prevention of disease.

As used herein, the term “probiotic bacterium” denotes a bacterium which ingested live in adequate quantities can exert beneficial effects on the human health. They are now widely used as a food additive for their health-promoting effects. Most of the probiotic bacteria are Lactic Acid Bacterium (LAB) and among them, strains of the genera Lactobacillus spp. and Bifidobacterium spp. are the most widely used probiotic bacteria.

In a preferred embodiment, the food-grade bacterium strain according to the invention is a Lactococcus lactis strain or a Lactobacillus casei strain or a Lactococcus lactis htrA strain (Poquet et al., Molecular Microbiology (2000) 35(5), 1042±1051) or Lactobacillus plantarum strain or a Lactobacillus rhamnosus or a Bifidobacterium longum strain or a Escherichia coli Nissle 1917 strain (Chen H et al. Mater Today Bio. 2023 Feb; 18: 100543).

In a preferred embodiment, the food-grade bacterium strain according to the invention is a Escherichia coli Nissle 1917 strain. Derived peptide of CDR3 Trypsin-3 neutralizing antibody of the invention:

The present inventors also demonstrated that the CDR3 of the Trypsin-3 neutralizing antibody of the invention (“NT3-7” and “NT3-12” sdAb) had the potential to inhibit the protease activity of Trypsin-3 and accordingly to be used as specific Trypsin-3 protease inhibitor (see below “The biological activities of the antibody of the invention using Protease activity assay”). Indeed, previous studies demonstrated that peptide derived from the CDR3 region of neutralizing antibodies, may also be used alone to neutralize the original therapeutic target in infectious field as in HIV therapy (see Dorfman T. et al THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 39, pp. 28529-28535, September 29, 2006; or Liu L et al JOURNAL OF VIROLOGY, Sept. 2011, p. 8467-8476 Vol. 85, No. 17).

Accordingly, the present invention also encompasses a polypeptide comprising

(a) the CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; and/or

(b) the CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 8.

A further object of the invention relates to a polypeptide comprising or consisting:

(a) the CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; and/or

(b) the CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO:

8, for use in therapy.

(a) the CDR3 of NT3-7 sdAb having a sequence set forth as SEQ ID NO: 4; and/or

(b) the CDR3 of NT3-12 sdAb having a sequence set forth as SEQ ID NO:

8, for use in the treatment of Trypsin-3 associated pathologies.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Inhibition of Trypsin-3 by NT3-7 and NT3-12.

To quantify the capacity of NT3-7 and NT3-12 to control Trypsin-3 activity, inhibition of Trypsin-3 activity are plotted with increased concentrations of single domain antibody. The half maximal inhibitory concentration (ICso) with a semi-log representation (a, b).

To determine the inhibition mode, initial reaction rates are plotted in which peptide substrate Z-GPR-pNA concentration and NT3 concentration were varied; fitted lines obtained from multiple regression are superposed on the data (c, d). All studies were performed with a final Trypsin-3 concentration of 0.5 nM and with substrate concentration ranging from 25 to 250 uM as indicated on the x axis.

All represented graphics are representative of 3 independent experiments. The apparent inhibition constant was calculated with a Lineweaver-Burk plot generated with these data (d).

Figure 2. Specificity of inhibition for selected single domain antibodies against Trypsin-3

Prior to added substrate, protease was pre-incubated for 15 min with single domain antibody with a ratio at I/E= 1000. NR single domain antibody: non relevant single domain antibody as negative control.

Figure 3. Points of attention for the bi-paratopic development.

Table described different possible combinations between Trypsin-3 inhibitors and the other SdAbs presenting high affinity for Trypsin-3 (a). Elisa sandwich assay to highlight potential interaction between two SdAbs on Trypsin-3; NT3.7 or NT3.12 was coated in wells (b). In silico analysis based on the 3D structure of Trypsin-3 to determine the longest lengths between two distant epitopes on the protease.

Figure 4. Bi-paratopic characterization.

Inhibition properties of bi-paratopic single domain antibodies compared to monoparatopic SdAbs against Trypsin-3 activity. The curves represent the percentage of resulting proteolytic activity according to the single domain antibody concentration (I, nM) added into the reaction mix (a).

Single cycle kinetics surface plasmon resonance measurements on Trypsin-3 captured on an Ni-NTA (Nickel affinity capture of 6xHis) chip (b). Bold line corresponds to the raw data measurements, and the fit curve is displayed in fine. Analytes was recombinant NT3-7/12 injected at increasing concentrations (1.5, 3.12, 6.25, 12.25 and 25 nM; arrow).

Figure 5. Effect of NT3 single domain antibodies on malignant growth of PC3 prostate cancer cells PC3 cells treated with 100 to 1000 nM NT3-7/12, NT3-7/7 (negative control regarding Trypsin-3 inhibition) or with buffer only (-) for 24H. After fixation and Phalloidin staining to delineate the cell’s surface, cellular circularity was measured. The index number equals 1 for a circular object and less than 1 for an object departs from circularity. Results are means and SEM for at least 200 characterized cells, (a).** p<0.002, *** p<0.0002, **** p<0.0001 for Two-way Anova with Turkey correction as post-test, p value comparison for indicated conditions. To assess the effect of inhibitor NT3 on migratory capacities of PC3 cells, cells were cultured in separate compartment for 24h and then, PC3 cells were treated with 25 to 1000 nM NT3-7/12, NT3-7/7 (negative control regarding Trypsin-3 inhibition) or with buffer only (-) for 14h. Images were taken immediately after the wound scratch (0 h) and every hour for 14h to monitor the process of cell migration into the gap. The gap closure rate was calculated and plotted on graph (b). ** p<0.002, *** p<0.0002 for one-way Anova with Turkey correction as post-test, p value comparison for untreated condition.

EXAMPLE 1: SELECTION OF VHHs TARGETING TRYPSIN-3

Materials and Methods

Plasmids

The previously described pAOT7 expression vector was used, in which any humanized synthetic single domain antibody (Nb) from the NaLi-Hl library can be inserted between Ncol and Notl restriction sites (Moutel, S., et al., NaLi-Hl : A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife, 2016.). This vector allowed the production of recombinant proteins in E.coli cytoplasm. A 6His-Myc- 6His tag downstream of the Notl site encodes the following translated sequence: H H H H H H GA AE Q K L I S E E D L N G G S P V GRH HH H H H * (SEQ ID NO: 22), thus producing pAOT7-Nb-6His-myc-6His form. The 6His-myc-6His tag downstream of the Notl site was replaced by a synthetic Flag-Ctag DNA fragment encoding the following translated sequence: A A A G G G S G GD Y K D D D D K G Y Q D Y E P E A *(SEQ ID NO: 23), thus producing pAOT7-Nb-Flag-Ctag form, as described (Keller, Tardy, analytical chemistry 2021). pCMV-IL2ss-Trypsin-2x-Strep-tag and pCMV-IL2ss-Trypsin-CBD-tag plasmids expression contain the IL2 signal sequence (IL2ss) that facilitates protein secretion. For pCMV plasmids amplification, ampicillin resistance assay was performed.

Trypsin- 1 and Trypsin-2 inactive proteases mutants were gene-synthesized as codon- optimized version of the human cDNA of pro-Trypsin-3 and SI 95 A pro-Trypsin-1,-2 and -3 downstream of the Interleukin-2 secretion signal sequence which allow efficient and homogenous secretion of the proteinases. The constructed were subcloned using Afllll and Notl restriction sites into 2 different pCMV plasmids, derived from pcDNA3.1 (thermo fisher), modified to allow expression of protein in fusion with a C-terminal purification tag, either a 2xStrep-tag® (similar to Twin-Strep-tag®from IBA-Lifesciences) or a Chitin Binding Domain.

Artificial gene synthesis of biparatopic single domain antibodies composed of two single domain antibodies sequences separated by a flexible linker sequence (GGGGSGGGGSAGSAAGSGEGGGGSGGGGSGGG SEQ ID NO: 21) were synthesized chemically (Genecust, Boynes, France), then sub-cloned in pAOT7-NcoI-NotI-FlagCtag digested empty plasmid, thus producing pAOT7-hs2dAbl-linker-hs2dAb2-FlagCtag. For all pAOT7 plasmids expression and purification, kanamycin resistance assay was performed using BL21(de3)pLysS strain (LI 195, Promega).

Expression and purification of Trypsins.

HEK293F were grown in serum-free Freestyle ™ 293 Expression Medium (#12338018, Thermofisher) without any antibiotic at 37°C in a humidified incubator with 8% CO2 and a rotation of 125rpm. This medium is used to grow, maintain and transfect cells. Cells were passed when they reached a density between lxlO^A6 and 3xlO^A6 viable cells and viability was determined using trypan blue. HEK293F cells were transfected with Freestyle MAX Reagent (16447100, Thermofisher), as indicated by the supplier.

Subtractive Phage Display Panning for isolating Trypsin-3 specific single domain antibodies

The NaLi-Hl library of humanized synthetic single domain antibody was used for this study. A subtractive panning protocol was designed to isolate Nb selective for Trypsin-3 mature conformation. The chitin binding domain (CBD) from chitinase Al or 2x-Strep-tag fusion of Trypsin- 1, Trypsin-2 and Trypsin-3 SI 95 A mutants and Trypsin-3 wt were captured freshly after cell lysis on magnetic beads before incubation with the library phages. Strep-Tactin® coated beads of Chitin magnetic beads were used alternatively for the capture of antigens for the three rounds of phage display. HEK293T (ATCC; Rockville, USA) were grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, life technology) supplemented with 10% of heat-inactivated FBS (Foetal Bovine Serum) (BioWittaker). The passages HEK293T were performed by PBS buffer, to avoid the use of Trypsin solution. The cells were transiently transfected by JetPrime reagent, as indicated by the supplier (PolyPlus Transfection), with plasmids which expressed IL2ss-pro-Trypsins-2S or IL2ss-pro-Trypsins-CBD. After 4 days of transfection the supernatant was recovered and incubated with magnetic MagStrep "type3" XT (IBA-Lifesciences, 2-4090-002) or Chitin Binding Domain coated beads (New England Biolabs, E8036) to capture and purify secreted Trypsins. For the first round of panning, phages were previously adsorbed on empty chitin (NEB) or MagStrep type 2 magnetic beads (IB A) to remove non-specific binders. From the second and the third round of panning, depletion steps on mutated Trypsin- 1 and mutated Trypsin-2 were included. The adequate amount of antigen- coated beads was incubated for Ih at 4°C on a rotative wheel with the phage library (10¹¹ phages diluted in ImL of TBSC+ 0.01% Tween + 0.5% casein). Phages and antigens-bound MagStrep- coated beads or Chitin beads were recovered on a magnet. Beads were washed with TBS-Tween 0.1% 10 times (round 1), 25 times (round 2).

Expression and purification of single domain antibodies

Cytosolic expression of Nb-6His-Myc-6His and of Nb-Flag-Ctag was performed in BL21(DE3)pLysS E.coli (#L1195, Promega) from the pAOT7 vector. Transformed bacteria cells were grown in a 500pL TB/kanamycin (35pg/mL) medium supplemented with 1% glucose and 3% ethanol in the start culture and incubated 2h at 37°C under 220rpm agitation. Cells were then grown in lOmL TB/kanamycin (35pg/mL) supplemented with 1% glucose and 3% ethanol overnight at 37° prior to dilution of the pre-culture in 2L-flasks containing 500mL of the same media with 1% glucose and 3% ethanol. Cells were allowed to grow at 37°C until OD600 reached 0.5-0.7, then diluted with 500mL of TB without glucose nor ethanol to reach a percentage of 0.5% glucose in the medium. When OD600 reached 0.5-0.7, cells were then induced with IPTG (Isopropyl P-D-l -thiogalactopyranoside, 18824P, Sigma Aldrich) at a final concentration of lOOpM and grown for an additional 16h at room temperature under agitation. Cells were harvested by centrifugation (30 min, 6000g, 4°C). After removing supernatant, pellets were snap frozen in liquid nitrogen and stored at -80°C before processing. The pellets were incubated at 37°C, resuspended in lOmL of ice-cold lysis buffer (20mM Tris, 150mM NaCl pH8, lx lysozyme). They were kept for 30min on ice and bacterial suspensions were sonicated on ice for 15 pulses with 10 s intervals. The sonicated solution was centrifuged (30 min, 12000g, 4°C) to separate pellet from supernatant containing soluble proteins. The supernatant was kept on ice and protein quantification was done by BCA assay (#23225, Thermofisher). Proteins were diluted to Img/mL with the correct ice-cold buffer and filtered with a 0.45pm filter.

Then, supernatants containing Nb were purified by affinity chromatography. The purification of Nb- 6His-Myc-6His was performed using Ni-NTA coated beads (Qiagen). The beads were washed with 2.5 mL of washing buffer (50mM Na2HPO4 pH8, 300 mM NaCl, lOmM imidazole). Single domain antibodies were then eluted with elution buffer (50mM Na2HPO4 pH7, 500mM NaCl, 300mM imidazole) and dialyzed thrice against PBS IX without CaC12 nor MgC12 for 16h at 4°C and then 2x2h at 4°C. Purity was assessed by SDS-PAGE followed by Coomassie Staining. After a BCA assay, glycerol was added to obtain 20% glycerol solutions. Aliquots were flash-freezed in liquid nitrogen and stored at -80°C.

The purification of Nb-FlagCtag was performed using a CS C-TAGXL MINICHROM 1 ML (Thermofisher) column and a CaptureSelect™ C-tag Affinity Matrix (Thermofisher). This matrix was equilibrated with 2CV of 20mM Tris, 150mM NaCl pH8 buffer at a flowrate of Img/mL. After injecting clarified lysate at a flowrate of Img/mL, the column was washed with 10CV of 20mM Tris, 150mM NaCl pH8 buffer at a flowrate of Img/mL. Finally, C- tagged-single domain antibodies were eluted in two isocratic steps: in 17% of 20mM Tris, 150mM NaCl pH8 in the presence of 2M MgC12 followed by 100% of the previous solution. Three consecutive dialysis against PBS IX without CaC12 nor MgC12 were then performed at 4°C, one overnight, the two others of 2h. Residual MgC12 has been shown to interfere with further enzymatic assays, and the quality of dialysis was tightly controlled. Purity was assessed by SDS-PAGE followed by Coomassie staining. After the three rounds of dialysis and BCA assay, glycerol was added to obtain 20% glycerol solutions. Aliquots were flash-freezed in liquid nitrogen and stored at -80°C.

Relative ELISA for evaluating antigen-binding activity

For the identification of single domain antibody detecting captured Trypsin, ELISA wells of Strep-Tactin-coated plates (IBA-Lifesciences®) were coated with 20 to 50 nM of recombinant mature Trypsins-2S at 4°C overnight and then blocked with 5% milk in TBSC- Tween 0.05% (blocking buffer) for Ih at RT. Single domain antibody diluted in blocking buffer were applied to the ELISA wells in duplicates for 2h30 at RT. The interaction between Trypsins and Single domain antibody was revealed by incubation with anti-myc-HRP (18824P, QED Bioscience) Ih at RT and the addition of 100 pL chromogenic substrate (Thermoscientific®, 1- step ultraTMB) for 5 to 30 min. The reaction was stopped with 50 pL H2SO4 IN and absorbance at 450 nm was measured using a FLUOstar OPTIMA microplate reader (BMG LABTECH, Ortenberg, Germany). Plates were washed one to three times with washing buffer (TBSC-Tween20 0.05%) after each step. All steps were performed under agitation (40 rpm).

For the capture or sandwich ELISA assays, 6His-tagged Single domain antibodies were diluted in TBS IX-lOmM CaC12- 0.05% Tween (TBSCT) to obtain a working concentration of 500nM, then coated according to the plate map on Nickel plate (15142, Life technologies) (lOOpL per well) and incubated Ih at RT under slow agitation. Each well was filled with lOOpL of active Trypsin-3-2S (200 nM). After three washes, each well was filled with lOOpL of Flag- tagged NT3s (NT3-1, NT3-3, NT3-7, NT3-12 and NT3-16) except the 2x-Streptag coating controls which were filled with TBSCT-5% milk alone. After rounds of washes, His coating controls were incubated with an anti-myc antibody (18824P, QED Bioscience, 1 : 10 000), Streptag coating controls were incubated with Streptactin-HRP (2-1502-001, IB A, 1 :500) and assay wells were incubated with an anti-Flag M2-HRP (A8592-2mg, Sigma Aldrich, 1 :20000) during Ih at RT under slow agitation. The ELISA reaction was visualized by the addition of 100 pL chromogenic substrate (1-step ultra TMB, Thermofisher) for 30 min and stopped with 50 pL H2SO4 IN. Finally, the optical density (OD) was measured at 450nm using a Varioskan Flash reader (Thermofisher).

Proteases activity

Trypsin-3 activity assays were performed using recombinant Human active Trypsin-3 (3714-SE-10, Bio-techne R&D) diluted in BOC-CHAPS-0.1% BSA Buffer (lOOmM Tris HC1, ImM CaC12, 0.1% BSA, pH8) at a final concentration of 0.5nM. It was incubated 15min at 37°C with each single domain antibody diluted in enzymatic buffer. The N-p-Tosyl-GPR- amino-4-methylcoumarin hydrochloride (0.1 mM) substrate was added after incubation. Substrate degradation was calculated by the change in fluorescence (ex: 355nm, em: 460nm), measured over 20 min at 37°C on a microplate reader Varioskan Flash (Thermofisher). The activity measurement of Thrombin (T6884, Sigma Aldrich), and Kallikrein 5 (1108-SE-010, Biotechne-R&D) was performed as described previously. Pro-kallikrein 1 (2337-SE-010, Bio- techne R&D) was activated by thermolysin (3097-ZN-020, Bio-techne R&D) treatment during Ih at 37°C in activation buffer (50 mM Tris, 10 mM CaC12, 150 mM NaCl, pH 7,5) and all activity assay was done in assay buffer (50 mM CHES, 250 mM NaCl, pH 10).

IC50 was calculated using GraphPad Prism software by plotting %inhibition versus log[I], To account for the effect of substrate KM on the inhibition constant, Ki was calculated using Lineweaver Burk plots.

Affinity Measurement

Single domain antibodies binding studies based on SPR (Surface Plasmon Resonance) technology was performed on a BIAcore T200 optical biosensor instrument (GE Healthcare). Capture of recombinant Nb-6xHis was performed on a nitrilotriacetic acid (NTA) sensor chip in HBS-P+ buffer (lOmM HEPES pH 7.5, 150mM NaCl and 0.05% surfactant P20). For immobilization strategies, flow cells were loaded with nickel solution (Sigma Aldrich) in order to saturate the NTA surface with Ni2+ and an extra wash using running buffer containing 3mM EDTA after the nickel injection. His-tagged Single domain antibodies were diluted in HBS-P+ buffer in flow cells at a concentration of 50pg/mL. As Single domain antibodies were smaller proteins than their Trypsin-3 target, Nb-6His were captured on the sensorchip at 4°C. Active Trypsin-3 or pro-Trypsin-3 were used as analytes. Analytes were injected sequentially at increased concentrations ranging between 9 nM to 500 nM in a single cycle without regeneration of the sensorchip between injections. Dilutions of the analytes were done in HBSC-P+ buffer (HBS-P, lOmM CaC12). The four flow cells (FC) of the sensorchip were used: one (FC1) to monitor nonspecific binding and to provide background corrections for analyses and the other three flow cells (FC2,3 and 4) containing immobilized Nb-6His for measurement. A non-relevant Nb was used as negative control (Bery, Cell Chem Biol 2019). FC1 sensorgrams were subtracted from sensorgrams obtained with immobilized Trypsin-3 to yield true binding responses. Kinetics constants (kon, koir, KD=k₀n/k₀ff) were calculated using BIAevaluation 4.0.1 software and the 1/lLangmuir binding model was chosen. This model determines the association constant (kon) and takes into account the dissociation occurring during the association phase. Therefore, the calculated values do not necessary correlate with apparent slope of the sensorgram.

PC3 treatments

PC3 cells (ATCCCRL-1435; provided by Dr Olivier Cuvillier, IPBS, Toulouse, France) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Fcetal Bovine Serum) (Life Technologies), IX Penicillin/streptomycin and IX non essential amino acids solution (Thermofisher #11140050) at 37°C in a humidified incubator with 5% CO2. For morphological assays, 10 000 cells by well containing glass coverslips were seeded. After 3 days, DMEM medium of the cells was removed and cells were treated for 24h. After the treatment, cells were washed once with PBS IX. Very few washes enabled the cells not to take off. Cells were fixed using paraformaldehyde 4% during lOmin and permeabilized with 0.2% Triton during lOmin. Coverslips were incubated for 30min at RT with a solution of 1/200 Alexa Fluor 647 Phalloidin (#A22287, Thermofisher) staining cellular membrane. Coverslips were shortly rinsed twice and mounted with ProLong Gold antifade reagent with DAPI (P36935, Invitrogen). Image J software was used to determine the roundness index of each cell. At least 200 cells were analyzed per condition and experiment was repeated more than 3 times.

In silica analysis

The in silico analysis to determine the linker length enabling a binding of two Nb together on Trypsin-3 was performed using Pymol 2.5.1 Software (Schrodinger, LLC) and a 3- D structure model of Trypsin-3 based on 2.38-A X-ray diffraction study (PDB 3L3T DOL 10.2210/pdb3L3T/pdb) (Salameh, M. A., Soares, A.S., Navaneetham, D., Sinha, D., Walsh, P.N., Radisky, E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin. (2010) J Biol Chem 285: 36884-36896). Nanobodies structures were modelled using Swiss model (https://swissmodel.expasy.org/) against 50 templates and generated structures were compared to the ones obtained with Alpha fold. This enabled us to validate the structures. All superpositions and structure figures were created using the graphics software PyMOL. The minimum length is based on the calculation of the maximum distance between two opposite epitopes of Trypsin-3. This enzyme presented an ovoid structure, the multiple distances were determined to estimate the appropriate length of the linker (Angstrom).

Statistical analysis

Analyses were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). Multiple comparisons were performed by repeated-measures one-way analysis of variance followed by Kruskal-Wallis procedure. Statistical significance was accepted at P < 0.05.

Results

Isolation of Trypsin-3 specific Single domain antibodies

To reach the goal of high selectivity towards Trypsin-3 we choose to perform a phage display selection using a highly diverse synthetic library of single domain antibodies (ref Moutel elife 2016). In order to avoid bias in diversity during the first steps of selection due to the protease activity of the antigen, and to obtain a large enrichment of Trypsin-3-reactive Single domain antibodies we produced recombinant mutated Trypsin- 1, Trypsin-2 and Trypsin- 3 as inactive mature protease and established a subtractive selection scheme. Accordingly, the phage displayed library was depleted on both inactive Trypsin- 1 and -2, followed by a positive selection on inactive Trypsin-3 in presence of an excess of inactive Trypsin- 1 and -2 during two rounds. The last round of selection was performed using wild type active Trypsin-3 in order to favor the enrichment of inhibitory single domain antibodies. Selected single domain antibodies were screened by ELISA assay to depict their detection of Trypsin- 1, Trypsin-2, pro- Trypsin-3, and active Trypsin-3. Sequencing 17 clones with apparent selectivity for active Trypsin-3 resulted in the identification of 5 different single domain antibodies (NT3-1, NT3-3, NT3-7, NT3-12, NT3-16). To confirm their selectivity and affinity towards active Tryspin-3, the 5 single domain antibodies were purified and further characterized. First, we performed a binding assay with 5 hits based on surface plasmon resonance (SPR) analysis (Table 6). The single domain antibodies were immobilized onto the sensorchip, their binding capacity was tested in real time with various injecting recombinant Trypsin-3 concentrations and analysis provided three parameters, association rate (&a), dissociation rate (kd), and dissociation constant (KD) at equilibrium. The 5 single domain antibodies bind to human matured Trypsin-3 wild type or catalytically inactive mutant, but no binding was observed with pro-form of Trypsin-3 (data not shown) or with mature Trypsin- 1 and -2 isoforms. We observed a typical binding pattern of SdAbs with very fast association and fast dissociation rates, with dose-dependent response. The five NT3 exhibited very interesting affinity for Trypsin-3 with KD value less than 10 nM. Despite a very high association constant, NT3-16 dissociated much more rapidly to the protease (Table 6). Stoichiometry of interaction between active Trypsin-3 and each single domain antibody was determined to be based on 1 : 1 molecule.

Characterization of Trypsin-3 Single domain antibodies as protease inhibitor

To assess the inhibition capacity of each single domain antibodies for Trypsin-3, we conducted enzyme inhibition experiments, monitoring cleavage of a fluorogenic peptide substrate by Trypsin-3 in the presence of varying concentrations of the single domain antibodies. In contrast to the binding assay, only NT3-7 and NT3-12 exhibited inhibition capacity of active Trypsin-3. To quantify the capacity ofNT3-7 andNT3-12 to control Trypsin- 3 activity, inhibition of Trypsin-3 activity was monitored with increased concentrations of single domain antibody. The half maximal inhibitory concentration (ICso) obtained with a semilog representation was in the low micromolar range (Figure 1). NT3-7 displayed a much lower ICso compared to NT3-12 (IC50 = 1.5± 0.3 and 8.81± 1.45 uM, respectively) (Figure 1 a-b).

To characterize the inhibition mode of single domain antibodies, competitive inhibition experiments were conducted using various concentrations of inhibitor and substrate to detect protease activity in the reaction mixture. The data generated from the progress curves were used to determine the mode of interaction between the inhibitor and Tryspin-3 (Figure 1 c-d). Presence of NT3-12 reduced the KM value whereas the Vmax remained similar with the protease alone condition. NT3-12 interacted with active Trypsin-3 as competitive inhibitor with an apparent Ki of 535 nM. In contrast, NT3.7 single domain antibody displayed a different mode of inhibition, it seems to counteract protease activity as a non-competitive inhibitor (change of KM and Vmax) with an apparent Ki of 46 nM.

The paratope of single-domain antibodies consists of three complementaritydetermining regions (CDRs) structured by framework residues. In the context of this study, the frameworks of all single-domain antibodies are identical; the diversity of paratopes is supported by the CDRs. When we superimpose the modeled structures of each strong inhibitor antibody (NT3-7 or NT3-12) with those either having (NT3-1, NT3-3, NT3-16) or lacking (NT3-2, NT3- 5) affinity for Trypsin-3, we observe that only the antibodies with affinity for Trypsin-3 have the same folding in CDR1 and CDR2. The capacity to inhibit Trypsin-3 appears to be dependent on the shape of the CDR3 loop (Data not shown).

Selectivity of single domain antibodies to inhibit other protease activities was then assessed against a panel of proteases closed to the trypsin-like activity (Trypsin- 1, Trypsin-2, Thrombin, KLK1, KLK3 and KLK5). Inhibition tests showed that at the molecular concentration of SdAbs which is able to inhibit 42% of Trypsin-3 activity (Inhibitor/Protease = 1000), both NT3s are poor inhibitor of other Trypsin-like proteases (inhibition max = 12% with KLK5). Addition of Nb into the reaction mix of Trypsin-1 induced a decrease of protease activity but this is not specific since addition of a non-relevant Nb also induced a reduction of protease activity and this is not Nb dose-dependent (data not shown). In the same way, NT3-7 and NT3-12 had no effect on KLK3 and Thrombin (Figure 2). Therefore, both single domain antibodies are very selective inhibitor of Trypsin-3.

Development of biparatopic Trypsin-3 single domain antibodies.

To increase the avidity of Trypsin-3 Single domain antibodies, we developed biparatopic dimers based on a Trypsin-3 inhibitor (NT3-7 or NT-12) genetically fused to one of the other single domain antibodies with high affinity for the Trypsin-3 (Figure 3A). The possibility of simultaneous interaction of both single domain antibodies on Trypsin-3 was firstly assessed by ELISA-sandwich assay. The Trypsin-3 inhibitor single domain antibody carrying a HIS tag was immobilized on the surface of microplate wells as a capture moiety, then incubated first with Trypsin-3 and further with the candidate flag-tagged single domain antibody as a detection moiety of Trypsin-3. After washing, the quantity of bound-single domain antibody was revealed using an anti-FLAG antibody coupled to HRP. In these conditions, only 3 Single domain antibodies were able to reveal Trypsin-3. NT3-1, NT3-12 and NT3-16 were able to bind on Trypsin-3 in the same time than NT3-7, and NT3-1 bound on protease with the competitor inhibitor NT3-12 (Figure 3B), indicating that biparatopic combination were possible.

To optimize the linker favoring the interaction between both single domain antibodies on Trypsin-3, we performed an in silico analysis based on Trypsin-3 model of the 3D-structure. Trypsin-3 is an ovoid protein, then the distance between the two farthest points has been calculated (data not shown). In addition, the sequence of the linker was designed to allow flexibility of the bioactive biparatopic molecule and avoiding amino acids known as predicted protease sensitive-site. We then empirically designed a linker of a potential length of 70.5 A mainly based on stretches of Glycine, and Serine or glutamate residues as gatekeeper of solubility (Chen, Zaro, & Shen, 2013) (data not shown). Five combinations were produced in heterologous system and tested for their inhibitory capacity. Surprisingly, the NT3-7 dimer as well as NT3-7+NT3-1 were not able to inhibit Trypsin-3 (data not shown). The NT3-7 fused to the highly affine NT3-16 behaved like a weak inhibitor (Figure 4A). In contrast, addition of both inhibitors (NT3-7+NT3-12) drastically increase the inhibition capacity of Nb (Figure 4A). The biparatopic single domain antibody had much lower ICso (0.043 pM) compared to the NT3-7 (ICso = 1.5 pM) and acted primarily as a competitive inhibitor of Trypsin-3. The other orientation of the fusion, i.e NT3-12 linked to the NT3-7, presented the same behaviors against Trypsin-3 (Figure 4A).

To complete the characterization of biparatopic Trypsin-3 inhibitor, its binding kinetics were evaluated by SPR. The KD value for the biparatopic NT3-7/12 was more than 30-fold lower than for the NT3-7 (Figure 4B, Table 6), 0.046 and 1.31 nM respectively, indicating a very high affinity of the NT3-7/12 for active Trypsin-3. The interactions between the targeted protease and the inhibitor have been drastically increased. As expected, the dissociation of the complex appeared very slow on the sensor chip (Figure 4B). In addition, the fusion between both Trypsin-3 inhibitor single domain antibodies also improved the selectivity of targeted proteases. The biparatopic Nb was only able to inhibit the active Trypsin-3 with a competitive mode, no more inhibition of KLK5 was observed (Data not shown).

In cellulo efficacy of biparatopic Trypsin-3 single domain antibody

To assess the capacity of NT3-7/12 to control the Trypsin-3 activity in a complex environment, we used PC3 cells. These cells displayed a native growth morphology characterized by “fibroblastic” shape. It has been shown that genetic ablation of Trypsin-3 expression in these cells induced a phenotypic change towards a rounded shape (Cohen et al., 2016). We used this trait, the circularity to evaluable the inhibitory capacity of single domain antibodies. PC3 were treated with different concentrations of NT3-7/12 or NT3-7/7, a non- inhibitory biparatopic Nb as negative control. Addition of NT3-7/7 into the culture medium of PC3 had no effect on cells whereas increased concentrations of NT3-7/12 induced a change of cellular shape, the elongated cells became round (Figure 5A). Dose effect on cellular circularity parameter observed with NT3-7/12 allowed us to calculate a median effect concentration (ECso) value at 642 nM. Therefore, the biparatopic Nb is able to tightly control the secreted Trypsin-3 activity in a challenging environment. Table 6: Kinetic parameters of interaction between Trypsin-3 and single domain antibodies

EXAMPLE 2

To assess the capacity of NT3-7 or NT3-7/12 to control the Trypsin-3 activity in a human context, colonic biopsy sections from IBS patients were pre-incubated with an anti- Trypsin-3 single domain antibody at 10 pM and in situ zymography was performed as described in Rolland-Fourcade et al, (Rolland -Fourcade et al. Epithelial expression and function of Trypsin-3 in irritable bowel syndrome. Gut 2017). The proteolytic activity detected at the level of epithelial cells in control condition was drastically decreased in the presence of the anti- Trypsin-3 single domain antibody. Therefore, the anti-Trypsin-3 single domain antibody is able to inhibit over-active Trypsin-3 released by human epithelial cells in IBS condition.

EXAMPLE 3

As Trypsin-3 was described to be crucial in the metastatic process (Hockla, 2012), capacity of NT3-7/12 to control cell migration was assessed on a wound healing assay with PC3 cells. NT3-7/12 was added to the culture medium in the same time than the gap was generated. NT3-7/12 inhibitor decrease the migration rate of PC3 in a dose dependant manner (Figure 5B). Addition of NT3-7/7 as no effect on PC3 migratory capacity demonstrating that the biparatopic Nb is able to inhibit the migration of metastatic prostate cancer cells based on control of Trypsin-3 activity. Table 7: Useful amino acid sequences for practicing the invention

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Claims

CLAIMS:

1. An anti-Trypsin-3 single domain antibody (sdAb), wherein said antibody specifically binds to human Trypsin-3 protein and does not bind to pro-form of Trypsin-3 and/or to mature Trypsin-2 and/or Trypsin- 1 isoform.

2. The anti-Trypsin-3 single domain antibody according to claim 1 wherein the single domain antibody has at least one or more of the following properties:

(i) it further exhibit inhibition capacity of active human Trypsin-3 (neutralizing antibody)

(ii) it binds to human active Trypsin-3 protein with a KD of 200 nM or below, 100 nM or below, 10 nM or below, 9 nM or below, 8 nM or below, 7 nM or below, 6 nM or below, 5 nM or below, 4 nM or below, 3 nM or below, 2 nM or below, 1 nM or below, 0.1 nM below, 0.05nM or less below.

3. The anti-Trypsin-3 single domain antibody according to claim 1 or 2, wherein said single domain antibody comprises

(b) a CDR1 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 6, a CDR2 of NT3-12 sdAb having a sequence set forth as SEQ ID NO: 7, and a CDR3 of NT3- 12 sdAb having a sequence set forth as SEQ ID NO: 8; or

(c) a CDR1 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 10, a CDR2 of NT3-1 sdAb having a sequence set forth as SEQ ID NO: 11, and a CDR3 of NT3- 1 sdAb having a sequence set forth as SEQ ID NO: 12; or

(d) a CDR1 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 14, a CDR2 of NT3-3 sdAb having a sequence set forth as SEQ ID NO: 15, and a CDR3 of NT3- 3 sdAb having a sequence set forth as SEQ ID NO: 16; or

4. The anti -Trypsin-3 single domain antibody according to any one of claim 1 to 3 wherein the single domain antibody comprises

(a) a variable heavy chain (VH) having at least 70% of identity with sequence set forth as SEQ ID NO: 1 (NT3-7); or

(b) a variable heavy chain (VH) having at least 70% of identity with sequence set forth as SEQ ID NO: 5 (NT3-12); or

(c) a variable heavy chain having at least 70% of identity with sequence set forth as SEQ ID NO: 9 (NT3-1); or

(d) a variable heavy chain having at least 70% of identity with sequence set forth as SEQ ID NO: 13 (NT3-3); or

(e) a variable heavy chain having at least 70% of identity with sequence set forth as SEQ ID NO: 17 (NT3-16).

5. The anti -Trypsin-3 single domain antibody according to any one of claim 1 to 4 wherein said single domain antibody has the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”); as SEQ ID NO: 5 (“NT3-12”) as SEQ ID NO: 9 (“NT3-1”) or as SEQ ID NO: 13 (“NT3-3”); or as SEQ ID NO: 17 (“NT3-16”).

6. The anti-Trypsin-3 single domain antibody according to claim 5 wherein said single domain antibody has the sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”) or as SEQ ID NO: 5 (“NT3-12”).

7. A cross-competing single-domain antibody which cross-competes for binding Trypsin- 3 with the single domain antibody according to any one of claims 1 to 5.

8. A polypeptide comprising at least one single-domain antibody according to any one of claims 1 to 5.

9. The polypeptide according to claim 8 which comprises at least two single-domain antibodies according to any one of claims 1 to 5.

10. The polypeptide according to claim 9, comprising two single-domain antibodies according to any one of claims 1 to 5.

11. The polypeptide according to any one of claim 8 to 10 which comprises a sequence of variable heavy chain (VH) set forth as SEQ ID NO: 1 (“NT3-7”) and a sequence of variable heavy chain (VH) set forth as SEQ ID NO: 5 (“NT3-12”).

12. A nucleic acid sequence encoding the anti-Trypsin-3 single domain antibody according to any of claims 1 to 6 or the cross-competing single-domain antibody according to claim 7 or a polypeptide according to any one of claim 8 to 11.

13. A vector comprising a nucleic acid sequence according to 12.

14. A pharmaceutical composition comprising the anti-Trypsin-3 neutralizing single domain antibody according to any of claims 1 to 6, or the cross-competing anti-Trypsin- 3 neutralizing single-domain antibody according to claim 7 and 2 or the polypeptide comprising at least one anti-Trypsin-3 neutralizing single-domain antibody according to any one of claim 8 to 11 or the nucleic acid sequence encoding an anti-Trypsin-3 neutralizing single domain antibody of claim 12 or the vector comprising a nucleic acid encoding an anti-Trypsin-3 neutralizing single domain antibody of claim 13.

15. A pharmaceutical composition, according to claims 14 for use as a drug.

16. The pharmaceutical composition according to claims 15 use for the treatment of gut disease associated with intestinal permeability selected from the list consisting of Irritable Bowel Syndrome (IBS), Inflammatory Bowel Diseases (IBD), celiac disease or pouchitis.

17. The pharmaceutical composition for use according to claim 15, for the treatment of cancer especially cancer associated with Trypsin-3.