Anti-tryptase-3 single domain antibody

Abstract

This invention relates to the generation and characterization of highly specific single-domain antibodies targeting trypsin-3, obtained from natural synthetic libraries using subtractive phage display technology. In this invention, the inventors disclose and generate single-domain antibodies (sdAbs) that specifically target trypsin-3 but do not recognize other serine proteases (trypsin-1 and trypsin-2) of the same family. Furthermore, utilizing the most promising sdAbs as structural units, the inventors designed and modified biepisode single-domain antibodies to identify highly selective binders capable of tightly inhibiting the target protease. These biepisode single-domain antibodies specifically inhibit trypsin-3 activity in the malignant proliferation of PC3 prostate cancer cells and in intestinal tissue sections from patients with irritable bowel syndrome (IBS), providing a new tool for anti-trypsin-3 immunotherapy. Therefore, this invention relates to single-domain antibodies that specifically bind to trypsin-3 protein but do not bind to other serine proteases (trypsin-1 and trypsin-2) of the same family. These specific antibodies can be used to detect trypsin-3, treat intestinal diseases related to intestinal permeability (irritable bowel syndrome, including gluten intolerance), and treat cancers, especially those related to trypsin-3.

Description

Technical Field

[0001] This invention relates to single-domain antibodies that specifically bind to trypsin-3 protein but not to other serine proteases in the same family (trypsin 1 and trypsin 2). These specific antibodies can be used to treat intestinal diseases related to intestinal permeability, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis, and can also be used to treat cancer. Specifically, this invention relates to single-domain antibodies that specifically target trypsin-3 for the treatment of irritable bowel syndrome (IBS), including gluten hypersensitivity, and for the treatment of cancers associated with trypsin-3. Background Technology

[0002] Proteases play crucial roles in physiological processes and disease manifestations. Although protease inhibitors have achieved some success in certain treatments, primarily for HIV infection, the number of approved protease inhibitors remains limited. In fact, one of the major challenges in developing protease inhibitors lies in achieving sufficient selectivity for the target active conformation among more than 550 human proteases (Puente, Sanchez, Overall, & Lopez-Otin, 2003). Proteases constitute a large family of enzymes with closely related homologs whose three-dimensional folded structures are similar, and therefore their active site conformations are also similar. For example, the active site residue sequences of members of the trypsin-like serine proteases family are highly homologous, and all described inhibitors cross-react with several conserved members (Otlewski, Jelen, Zakrzewska, & Oleksy, 2005). However, selective inhibition of serine proteases appears crucial because off-target effects can lead to serious disease (Puentee et al., 2003).

[0003] Human trypsin-3 is a unique digestive serine protease specifically designed to degrade trypsin inhibitors (Szmola, Kukor, & Sahin-Toth, 2003). The three main forms of digestive trypsin (anionic, meso, and cationic trypsin) show high sequence homology, with trypsin-3 differing from trypsin-1 by only 28 amino acid residues. The unique conformation of trypsin-3 is the result of an evolutionary mutation in the PRSS3 gene encoding trypsin-3: Gly 198 Replaced by Arg (Gly) 193 (Replaces trypsinogen form). Arg 193One of the most critical differences of trypsin-3 is that it is located at the active site, a position where a glycine residue is highly conserved in almost all other trypsin and serine protease members of the chymotrypsin family. Other unique trypsin-3 residues include Lys... 74 and Asp 97 Located on the periphery of the active site, this structure contributes to trypsin-3's resistance to typical trypsin inhibitors. These differences between trypsin-3 and other trypsins contribute to the formation of an unusually strong positive charge buildup around the primary specific pocket of trypsin-3 (Katona, Berglund, Hajdu, Graf, & Szilagyi, 2002). These distinguishable structural features undoubtedly affect the binding of the molecular spouse and can enhance potential inhibitory selectivity.

[0004] Although proteases exhibit high similarity at their catalytic sites, the loop regions surrounding the active site can confer significant structural diversity, leading to slightly different but significantly distinct three-dimensional structural arrangements (Goettig, Brandstetter, & Magdolen, 2019). Therefore, to screen for highly selective inhibitors, the binding interface with the target should not only cover the catalytic domain but also involve allosteric sites. Compared to small molecules, peptides are more likely to bind to both allosteric and active sites simultaneously, thus exhibiting higher selectivity. Antibodies, with their excellent affinity and selectivity, theoretically possess the ability to distinguish the binding of closely related members of the protease family. Furthermore, antibody technology has the unique advantage of targeting regions of proteases other than the natural inhibitor binding site (i.e., the active site region), thus enabling the development of allosteric inhibitors. Based on this, we developed a strategy to screen for single-domain antibodies (sdAbs) that can efficiently and selectively inhibit human trypsin-3. Single-domain antibodies constitute a highly promising tool, known for their high resistance to protease degradation (Asaadi, Jouneghani, Janani, & Rahbarizadeh, 2021), ease of modification, and ability to be expressed in bacteria. More interestingly, several single-domain antibodies (sdAbs) have been reported to bind to concave surfaces of antigens, such as the active site or loop region of an enzyme (Muyldermans, 2021).

[0005] Here, the inventors identified SdAbs targeting human trypsin-3 using a subtractive phage display strategy. Using the most promising SdAbs as building blocks, bipolar single-domain antibodies were constructed to obtain highly selective binders capable of tightly inhibiting the target protease. Summary of the Invention

[0006] The present invention provides an isolated antitrypsin-3 single-domain antibody, wherein the antibody specifically binds to human trypsin-3 protein.

[0007] In one specific embodiment, the antibody of the present invention has at least one or more of the following properties: (i) Not bound to trypsin-3 precursors or mature trypsin-2 or trypsin-1 isoforms, and / or; (ii) Has inhibitory ability against active human trypsin-3 (neutralizing antibody), and / or; (iii) The KD value of its binding to human active trypsin-3 protein is 200 nM or less, 100 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.05 nM or less, 0.1 nM or less, or 0.05 nM or less.

[0008] The present invention also relates to polypeptides comprising at least one single-domain antibody of the present invention.

[0009] The present invention further relates to the antitrypsin-3 single-domain antibody of the present invention, and polypeptides comprising at least one single-domain antibody of the present invention for the treatment of intestinal diseases associated with intestinal permeability, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis.

[0010] The present invention further relates to the antitrypsin-3 single-domain antibody of the present invention, and a polypeptide comprising at least one single-domain antibody of the present invention, for the treatment of cancer, particularly cancers associated with trypsin-3.

[0011] In a preferred embodiment, the cancer is a tumor associated with trypsin-3 activity. Invention Details In this invention, the inventors disclose single-domain antibodies (sdAbs) (also known as VHHs or single-domain antibodies) that specifically target trypsin-3 without targeting other serine proteases (trypsin-1 and trypsin-2) of the same family. The inventors generated and screened anti-trypsin-3 single-domain antibodies using a highly diverse synthetic single-domain antibody library via phage display technology (Moutel et al., 2016). Because this approach involves screening single-domain antibodies in vitro against the active form of the enzyme, the developed single-domain antibodies have the inherent advantage of recognizing the topology of 3-D epitopes and enzyme active sites. Current data reflect strong conformational selectivity for only one form of the protease (the mature active form of trypsin-3), as no interactions were detected between NT3 and other members of the trypsin-like family, as well as the precursor form of the target protease. Furthermore, the inventors constructed bipolar single-domain antibodies using the most promising sdAbs as structural units, screening for an inhibitor capable of highly selectively binding to and potently inhibiting the target protease. This bipolar single-domain antibody specifically inhibits the malignant growth of PC3 prostate cancer cells and the activity of trypsin-3 in intestinal tissue sections from IBS patients, providing a new tool for anti-trypsin-3 immunotherapy.

[0013] Antibodies according to the present invention This invention provides an anti-trypsin-3 single-domain antibody (sdAb) wherein the antibody specifically binds to human trypsin-3 protein. In some embodiments, this invention provides an anti-trypsin-3 single-domain antibody (sdAb) wherein the antibody specifically binds to human trypsin-3 protein and does not bind to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms.

[0014] The single-domain antibodies of the present invention preferably exhibit one or more additional desired functional properties selected from the group consisting of: (i) Not bound to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms, and / or; (ii) It has inhibitory ability against active human trypsin 3 protein (neutralizing antibody), and / or; (iii) The KD binding to human active trypsin-3 protein is 200 nM or less, 100 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, or 0.05 nM or less.

[0015] As used herein, the term "single-domain antibody" has its general meaning in the art, referring to a single-heavy variable domain of an antibody type that is naturally lacking a light chain and is found in camel mammals. Such single-domain antibodies are also known as VHHs or "single-domain antibodies" or "nanobodies®". For a general description of (single)domain antibodies, see also the prior art cited above, as well as EP 0368684, (Ward, Gussow, Griffiths, Jones, & Winter, 1989), (Holt, Herring, Jespers, Woolven, & Tomlinson, 2003), and WO06 / 030220, WO06 / 003388. VHHs have a molecular weight approximately one-tenth that of human IgG molecules and a physical diameter of only a few nanometers. One consequence of their small size is that single-domain antibodies (or VHHs) can bind to antigenic sites that are functionally unrecognizable to larger antibody proteins. This means single-domain antibodies (or VHHs) can serve as reagents for detecting occult antigens that are difficult to identify using classical immunological techniques, and can also be used as potential therapeutic agents. Therefore, another advantage of their small size is that single-domain antibodies (or VHHs) can inhibit the activity / interaction of target proteins by binding to specific sites in grooves or narrow crevices, thus functionally resembling classical small-molecule drugs rather than classical antibodies. Their low molecular weight and compact folded structure result in extremely high thermal stability, stability against extreme pH and proteolytic digestion, and lower antigenicity due to the lack of an Fc fragment. Another advantage of VHHs is their ease of movement from the circulatory system into tissues and their higher likelihood of crossing the blood-brain barrier, potentially treating conditions affecting nerve 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 low antigenicity to humans, indicate significant therapeutic potential. The amino acid sequence and structure of a single-domain antibody can be considered to consist of four frame regions, or “FRs,” referred to in the art and herein as “frame region 1” or “FR1”; “frame region 2” or “FR2”; “frame region 3” or “FR3”; and “frame region 4” or “FR4”; these frame regions are separated by three complementarity-determining regions, or “CDRs,” referred to in the art as “complementarity-determining region” or “CDR1”; “complementarity-determining region 2” or “CDR2”; and “complementarity-determining region 3” or “CDR3”, respectively. Therefore, a single-domain antibody can be defined as having the following general structure of amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively.In the context of this invention, the amino acid residues of the single-domain antibody are numbered according to the general numbering rules for the VH (heavy chain variable region) domain given by the International Immunogenetic Information System (http: / / imgt.org / ).

[0016] The term "trypsin-3," also known as "intermediate trypsin" or "TRY3," refers to trypsin-3 (EC 3.4.21.4), a serine protease encoded by the PRSS3 gene in humans. In humans, three serine protease (PRSS) genes encode trypsinogen: PRSS1 encodes trypsinogen-1 (cationic trypsin), PRSS2 encodes trypsinogen-2 (anionic trypsin), and PRSS3 encodes trypsinogen-3, of which at least two isoforms with overlapping mature peptide sequences (formerly known as intermediate trypsinogen and trypsinogen IV) have been functionally characterized. The mature protein of the PRSS3 gene uses the nomenclature for trypsin-3 proteins, which applies 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 for peptide bonds involving carboxyl groups of lysine or arginine. Four transcript variants encoding different isoforms of this gene have been reported. The full sequence of the human PRSS3 gene (the gene encoding trypsin-3) is known as Gene ID: 5646.

[0017] The protein sequences of the human trypsin-3 and its isotypes can be found in the NCBI database, with accession numbers as follows: Trypsin-3 variant 1 mRNA: NM_007343, and protein id: NP_031369 (isotype 1), (trypsinogen 4). Trypsin-3 variant 2 mRNA: NM_002771 and protein id: NP_002762 (isotype 2) (trypsinogen 3, intermediate trypsinogen); Trypsin-3 variant 3 mRNA: NM_001197097, and protein id: NP_001184026 (isotype 3), Trypsin-3 variant 4 mRNA: NM_001197098, and protein id: NP_001184027 (isotype 4) (trypsinogen 5).

[0018] The inventors identified PRSS3 variant 1 as the major transcript expressed in intestinal epithelial cells and colon tissue samples. However, five different transcripts encode the same active form of trypsin-3 protein (P35030|TRY3_human trypsin-3, P35030-2|TRY3_human isotype 2 of trypsin-3, P35030-3|TRY3_human isotype 3 of trypsin-3, P35030-4|TRY3_human isotype 4 of trypsin-3, and P35030-5|TRY3_human isotype 5 of trypsin-3).

[0019] An example of the amino acid sequence of human trypsin-3 (mature active form) is shown in SEQ ID NO: 29.

[0020] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple sites via weak non-covalent forces; the more interactions, the stronger the affinity. Affinity can be determined by measuring KD. As used herein, the term KD refers to the dissociation constant, which is derived from K... off With K on The ratio (i.e., K) off / K on The KD value of the antibody is obtained and expressed as molar concentration (M). The KD value of the antibody can be determined using methods recognized in the art, one of which is detection using surface plasmon resonance technology via a biosensor system such as the Biacore® system.

[0021] In one specific embodiment, the affinity of the antibody of the present invention for human trypsin-3 protein refers to the KD value of the antibody having 200 nM or less, 100 nM or less, preferably at least 10 nM or less, 9 nM or less, 8 nM or less, more preferably 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, at least 1 nM or less, and even more preferably 0.1 nM or less, or 0.05 nM or less (for bispecific antibodies), which is determined by surface plasmon resonance (SPR) technology in the “Affinity Measurement” section detailed in the examples below.

[0022] In some implementations, the single-domain antibody is a "humanized" single-domain antibody.

[0023] As used herein, the term "humanization" refers to the single-domain antibody of the present invention, wherein the amino acid sequence corresponding to the naturally occurring VHH domain has been "humanized," i.e., by replacing one or more amino acid residues in the naturally occurring VHH (particularly the framework sequence) amino acid sequence with one or more amino acid residues present at the corresponding site in the heavy chain variable region (VH) of a humanized conventional heavy chain antibody. Methods for humanizing single-domain antibodies are well known in the art. Generally, the selection of humanization substitution sites should ensure that the modified humanized single-domain antibody retains the advantageous properties of the single-domain antibody of the present invention. Those skilled in the art can identify and select suitable humanization substitution sites or combinations thereof. For example, the single-domain antibody of the present invention can be appropriately humanized at any framework residue shown in Table 7, provided that the modified single-domain antibody remains soluble and does not significantly lose its affinity for trypsin-3.

[0024] Single-domain antibodies (NT3-X) and derivatives In particular, the inventors have developed five fully human single-domain antibodies targeting human trypsin-3 (also referred to herein as NT3-1, NT3-3, NT3-7, NT3-12, and NT3-16). These single-domain antibodies have been screened to specifically and with high affinity target human active trypsin-3 (K). D The antibodies are available in concentrations of ~10 nM or less, ~7 nM or less, ~5 nM or less, ~3 nM or less, and ~2 nM or less, without binding to other serine proteases in the same family (trypsin-1 and trypsin-2), or to the precursor of trypsin-3 polypeptide. Furthermore, the inventors characterized some of these antibodies, confirming them as specific trypsin-3 protease inhibitors (NT3-7, NT3-12).

[0025] Therefore, in some embodiments, the single-domain antibody of the present invention is a trypsin-3 neutralizing single-domain antibody. Such antibodies are capable of inhibiting the protease activity of trypsin-3 (see below, “Evaluating the biological activity of the antibodies of the present invention using a protease activity assay”).

[0026] As used herein, a “neutralizing antibody” refers to an antibody (e.g., a single-domain antibody) capable of binding to and inhibiting the active form of trypsin-3 protein and improving intestinal permeability and pain observed in intestinal diseases associated with this damage, such as irritable bowel syndrome (IBS) including gluten hypersensitivity. In one specific embodiment, a neutralizing antibody refers to an antibody with an IC50 of at least 100 μM or less, preferably at least 50 μM or less, more preferably at least 10 μM or less, and even more preferably at least 1 μM or less, as determined by the protease activity assay described in the “Characteristics of Trypsin-3 Single-Domain Antibodies as Protease Inhibitors” section detailed in the examples below.

[0027] In some embodiments, the isolated antitrypsin-3 single-domain antibody according to the present invention comprises: (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0028] In one specific embodiment, the present invention relates to an antitrypsin-3 single-domain antibody, wherein the single-domain antibody comprises: The heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 1 (NT3-7); or The heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 5 (NT3-12); or The heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 9 (NT3-1); or The heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 13 (NT3-3); or The heavy chain variable region (VH) has at least 70% identity with the sequence shown in SEQ ID NO: 17 (NT3-16).

[0029] According to the present invention, a first amino acid sequence having at least 70% identity with a second amino acid sequence means that the first sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 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).

[0030] In one specific embodiment, the isolated single-domain antibody of the present invention has a heavy chain variable region (VH) sequence as shown in SEQ ID NO: 1 (“NT3-7”), SEQ ID NO: 5 (“NT3-12”), SEQ ID NO: 9 (“NT3-1”), or SEQ ID NO: 13 (“NT3-3”), or SEQ ID NO: 17 (“NT3-16”).

[0031] In one specific embodiment, the above-described single-domain antibodies bind to the same antigen and have the same or improved properties as the single-domain antibodies of the present invention (see specific trypsin-3 conjugates and / or specific protease inhibitor activities). The single-domain antibodies of the present invention are: antibodies having the CDRs shown in SEQ ID NO: 2 to 4 (“NT3-7”); antibodies having the CDRs shown in SEQ ID NO: 6 to 8 (“NT3-12”); antibodies having the CDRs shown in SEQ ID NO: 10 to 12 (“NT3-1”); antibodies having the CDRs shown in SEQ ID NO: 14 to 16 (“NT3-3”); and antibodies having the CDRs shown in SEQ ID NO: 18 to 20 (“NT3-16”).

[0032] Table 1 below describes the sequences of the heavy chain variable region (VH) and CDRs (or FRs) of the single-domain antibody NT3-7.

[0033] Table 1

[0034] Table 2 below describes the sequences of the heavy chain variable region (VH) and CDRs domain of the single-domain antibody NT3-12.

[0035] Table 2

[0036] Table 3 below describes the sequences of the heavy chain variable region (VH) and CDRs (or FRs) domains of the single-domain antibody NT3-1.

[0037] Table 3

[0038] Table 4 below describes the sequences of the heavy chain variable region (VH) and CDRs (or FRs) domains of the single-domain antibody NT3-3.

[0039] Table 4

[0040] Table 5 below describes the sequences of the heavy chain variable region (VH) and CDRs (or FRs) domains of the single-domain antibody NT3-16.

[0041] Table 5

[0042] Functional variant NT3-X Therefore, the present invention provides antibodies comprising functional variants of a VH region, wherein the VH region includes FRs and / or one or more CDRs of the single-domain antibody of the present invention. The functional variants of the VH (FR or CDR) used in the context of the single-domain antibody of the present invention still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or higher) of the affinity / affinity and / or specificity / selectivity of the parent antibody (i.e., a single-domain antibody (sdAb) NT3-X selected from NT3-1, NT3-3, NT3-7, NT3-12 and NT3-1), and in some cases, the affinity, selectivity and / or specificity of such a single-domain antibody of the present invention may be higher than that of the parent single-domain antibody (or VHH). These variants can be obtained through various affinity maturation protocols, including mutant CDRs (Yang et al., 1995), strand shuffling (Marks et al., 1992), using mutant strains of *E. coli* (Low, Holliger, & Winter, 1996), DNA shuffling (Patten, Howard, & Stemmer, 1997), and phage display (Thompson et al., (1996) and sexual PCR (Crameri, Raillard, Bermudez, & Stemmer, 1998). These functional variants typically retain significant sequence identity with the parental single-domain antibody (or VHH). The CDR sequence of the variant can differ primarily from the CDR sequence of the parental antibody sequence through conserved substitutions; for example, 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 conserved amino acid residue substitutions. The CDR sequence of the variant can differ primarily from the CDR sequence of the parental antibody sequence through conserved substitutions; for example, at least three, such as at least two or one, substitutions in the variant are conserved amino acid residue substitutions. In the context of this invention, conserved substitution can be defined as substitution within the following categories of amino acids: Aliphatic residues I, L, V and M Cycloalkenyl-related 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 Minimal residues A, G, and S The residues involved in the turn formation are 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 substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. The variant CDRs also largely retain conservation in hydrophobicity / hydrophilicity and residue molecular weight / size compared to the CDRs of the single-domain antibodies of this invention. The importance of the hydrophobic amino acid index in conferring biological functions of protein interactions is generally understood in the art. It is recognized that the relative hydrophobicity of amino acids determines the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid was assigned a hydrophobicity index based on its hydrophobicity and charge characteristics, as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also be measured, or alternatively, by a similarity score, as determined by using a BLAST procedure (e.g., using the standard settings BLOSUM62, open gap = 1 and gap extension = 1, BLAST 2.2.8, available via NCBI). Suitable variants typically exhibit at least about 70% identity with the parent protein. According to the invention, at least 70% identity between the first and second amino acid sequences means that the first and second sequences have 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% identity. According to the present invention, the first amino acid sequence and the second amino acid sequence have at least 90% identity, meaning that the first sequence and the second amino acid sequence have 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity.

[0043] In some embodiments, the single-domain antibody of the present invention is a single-domain antibody having a heavy chain variable region comprising (i) VH-CDR1 having at least 3, 2, or 1 conserved substitutions within the VH-CDR1 sequence of the single-domain antibody NT3-X (SEQ ID NO: 2 or SEQ ID NO: 6 or SEQ ID NO: 10 or SEQ ID NO: 14 or SEQ ID NO: 18); (ii) VH-CDR2 having at least 3, 2, or 1 conserved substitutions within the VH-CDR2 sequence of the single-domain antibody NT3-X (SEQ ID NO: 3 or SEQ ID NO: 7 or SEQ ID NO: 11 or SEQ ID NO: 15 or SEQ ID NO: 19); and (iii) VH-CDR3 having at least 3, 2, or 1 conserved substitutions within the VH-CDR2 sequence of the single-domain antibody NT3-X (SEQ ID NO: 4 or SEQ ID NO: 8 or SEQ ID NO: 19). SEQ ID NO: 12 or SEQ ID NO: 16 or SEQ The VH-CDR3 sequence of ID NO: 20 contains There are at least 3, 2, and 1 conservative substitutions.

[0044] As used herein, “NT3 analog” or “NT3 derivative” refers to a single-domain antibody that exhibits at least the same or better specific binding to trypsin-3 protein and has at least one biological activity of the single-domain antibody NT3 shown in SEQ ID NO: 1 (“NT3-7”); or SEQ ID NO: 5 (“NT3-12”); or SEQ ID NO: 9 (“NT3-1”); or SEQ ID NO: 13 (“NT3-3”); or SEQ ID NO: 17 (“NT3-16”). The NT3 analog may, for example, demonstrate the ability to inhibit active trypsin-3 protein experimentally (see Example 1: Materials and Methods / Protein Activity). In short, trypsin-3 activity can be measured using active human trypsin-3 by incubating the NT3 analog. After incubation, a trypsin-3 substrate (e.g., Np-toluenesulfonyl-GPR-amino-4-methylcoumarin hydrochloride) is added, and substrate degradation is calculated by changes in fluorescence on a microplate reader.

[0045] As described above, the biological activity of the antibodies of the present invention is, for example, reducing the level of trypsin-3 proteolytic activity. Assessment of trypsin-3 activity levels allows for the determination of the therapeutic properties of the single-domain antibodies (neutralizing antibodies) of the present invention, such as correction of intestinal permeability and pain observed in intestinal diseases associated with this damage, for example in irritable bowel syndrome (IBS) involving gluten hypersensitivity.

[0046] The specific binding of the antibody can be determined by any method known in the art. Many different competitive binding assays can be used for epitope binding. Immunoassays that can be used include, but are not limited to, competitive assay systems using techniques such as Western blotting, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, precipitin assay, gel diffusion precipitin assay, immunoradioassay, fluorescence immunoassay, protein A immunoassay, and complement fixation assay. Such assays are routine and well known in the art (see, for example, Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). For example, BIACORE® (GE Healthcare, Piscataway, NJ) is one of many routine forms of surface plasmon resonance analysis used for monoclonal antibody epitope clustering. Furthermore, routine cross-blocking assays can be performed, as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.

[0047] Single-domain antibodies competing with the single-domain antibodies of this invention Another aspect of the invention relates to a cross-competitive single-domain antibody that cross-competes with the single-domain antibody of the present invention to bind trypsin-3. In some embodiments, the cross-competitive single-domain antibody of the present invention cross-competes with a single-domain antibody comprising: (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0048] In some embodiments, the cross-competitive single-domain antibody of the present invention cross-competitively binds trypsin-3 with single-domain antibodies comprising or consisting of the following variable heavy chain (VH) sequences: SEQ ID NO: 1 (“NT3-7”), SEQ ID NO: 5 (“NT3-12”), SEQ ID NO: 9 (“NT3-1”), or SEQ ID NO: 13 (“NT3-3”), or SEQ ID NO: 17 (“NT3-16”).

[0049] As used herein, the term "cross-competition" refers to the property of single-domain antibodies to bind to the same specific region of an antigen. In this disclosure, a single-domain antibody that engages in "cross-competition" is an antibody that interferes with the binding of another single-domain antibody to an antigen in a standard competitive binding assay. According to a non-limiting theory, such a single-domain antibody may bind to the same, related, or adjacent (e.g., structurally similar or spatially close) epitopes as its competing single-domain antibody. Cross-competition exists if single-domain antibody A reduces the binding of single-domain antibody B by at least 60%, particularly at least 70%, more particularly at least 80%, compared to a positive control lacking one of the said single-domain antibodies, and vice versa. As those skilled in the art will understand, competition can be assessed in different assay settings. A suitable assay involves using BIAcore technology (e.g., using a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology. Another method for assessing cross-competition uses an ELISA-based approach. In addition, a high-throughput method for “binding” antibodies based on cross-competition is described in international patent application number WO2003 / 48731.

[0050] According to the present invention, the cross-competitive antibody described above retains the activity of the single-domain antibody, wherein the single-domain antibody comprises... (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0051] According to the present invention, the cross-competitive antibody described above retains the activity of a single-domain antibody comprising or consisting of the following variable heavy chain (VH) sequences: SEQ ID NO: 1 (“NT3-7”), SEQ ID NO: 5 (“NT3-12”), SEQ ID NO: 9 (“NT3-1”), or SEQ ID NO: 13 (“NT3-3”), or SEQ ID NO: 17 (“NT3-16”).

[0052] Therefore, in some embodiments, the cross-competitive single-domain antibody of the present invention is a trypsin-3 neutralizing single-domain antibody that inhibits the protease activity of trypsin-3.

[0053] In some embodiments, the cross-competitive single-domain antibody of the present invention binds to human trypsin-3 protein and does not bind to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms.

[0054] Single-domain antibody peptides and derivatives Single specific Another aspect of the present invention relates to a polypeptide comprising at least one single-domain antibody of the present invention.

[0055] Typically, the polypeptides of this invention comprise a single-domain antibody of this invention, which is fused to at least one additional amino acid sequence at its N-terminus, C-terminus, or both the N-terminus and C-terminus, thus forming a fusion protein. According to this invention, a polypeptide comprising only one single-domain antibody is referred to herein as a "monovalent" polypeptide. A polypeptide comprising or substantially composed of two or more single-domain antibodies of this invention is referred to herein as a "multivalent" polypeptide.

[0056] In some embodiments, the two or more single-domain antibodies (“multivalent” polypeptides) of the present invention can be directly linked (i.e. without using a adapter) or linked to each other via a adapter.

[0057] The linker is typically a linker peptide, and according to the invention, the linker peptide is selected to enable the two single-domain antibodies to bind to the same epitopes of two different trypsin-3 proteins. A suitable linker depends particularly on the epitopes, especially the distance between the epitopes on the two different trypsin-3 proteins to which the single-domain antibody binds; the selection of such linkers can be readily determined by those skilled in the art based on the disclosure herein after limited routine experiments. Furthermore, two single-domain antibodies binding different trypsin-3 proteins can also be interconnected by a third single-domain antibody (where the two single-domain antibodies can be directly linked or linked to a third domain antibody via a suitable linker). For example, such a third single-domain antibody can be a single-domain antibody that provides an extended half-life. For example, the latter single-domain antibody can be a single-domain antibody capable of binding (human) serum proteins such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more single-domain antibodies binding different trypsin-3 proteins are linked in tandem (directly or via a suitable linker), and a third (single) single-domain antibody (which can provide an extended half-life, as described above) is directly or via a linker linked to one of the two or more of the aforementioned single-domain antibodies.

[0058] This document describes suitable linkers associated with specific peptides of the present invention and may—for example, but not limited to—comprising an amino acid sequence preferably having 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 rather a convenient choice, for example, regarding the biopharmaceutical production of such peptides. The linker sequence may be a naturally occurring or non-natural sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in subjects administering the antitrypsin-3 protein peptide of the present invention. A useful set of linker sequences is that derived from the hinge region of heavy chain antibodies as described in WO96 / 34103 and WO94 / 04678. Other examples are polyalanine linker sequences, such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly / Ser linkers of varying lengths, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0059] According to one specific embodiment, at least two single-domain antibodies (“monospecific multivalent” polypeptides) of the present invention are linked by a linker derived from the hinge region of a heavy chain antibody. Such polypeptides are also referred to as “microantibodies.” The term “microantibody” corresponds to an antibody form containing a CH3 domain (from classical Ig) comprising an Fc fragment, followed by a hinge sequence fused to a VHH (or ScFv domain). Examples of “microantibodies” with ScFv domains are described in ((Hu et al., 1996; Kim et al., 2014; Nunez-Prado et al., 2015) and WO 94 / 04678).

[0060] According to the present invention, the single-domain antibodies and peptides of the present invention can be generated by conventional automated peptide synthesis methods or by recombinant expression. The general principles of protein design and preparation are well known to those skilled in the art. The single-domain antibodies and peptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to 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 peptides of the present invention can also be synthesized using exemplary peptide synthesizers via solid-phase techniques, such as the Model 433A of Applied Biosystems Inc. Any target protein generated by automated peptide synthesis or by recombinant methods can be determined using reversed-phase HPLC analysis. The chemical authenticity of each peptide can be determined by any method well known to those skilled in the art. As an alternative to automated peptide synthesis, recombinant DNA technology can be employed, in which a nucleotide sequence encoding a selected protein is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described below. Recombinant methods are particularly preferred for producing longer peptides.

[0061] Multispecific In some embodiments, the polypeptide comprises at least one single-domain antibody of the present invention and at least one other binding unit (i.e., targeting another epitope, antigen, target, protein, or polypeptide), which is typically also a single-domain antibody. Such polypeptides are referred to herein as “multispecific” polypeptides; in contrast, polypeptides comprising the same single-domain antibody are “single-specific” polypeptides. Therefore, in some embodiments, the polypeptides of the present invention may also provide at least one additional binding site targeting any desired protein, polypeptide, antigen, antigenic determinant, or epitope. This binding site may target the same protein, polypeptide, antigen, antigenic determinant, or epitope targeted by the single-domain antibody of the present invention, or it may target a different protein, polypeptide, antigen, antigenic determinant, or epitope than the single-domain antibody of the present invention.

[0062] Typically, one or more additional binding sites may comprise one or more portions, fragments, or domains of conventional chain antibodies (particularly human antibodies) and / or heavy chain antibodies. For example, the single-domain antibody of the present invention may optionally be linked to a conventional (typically human) VH or VL via a linker sequence.

[0063] The "bispecific" polypeptide of the present invention is a polypeptide comprising at least one single-domain antibody against a first antigen (i.e., trypsin-3 protein) and at least one additional binding site against a second antigen (i.e., different from trypsin-3 protein), while the "trispecific" polypeptide of the present invention is a polypeptide comprising at least one single-domain antibody against a first antigen (i.e., trypsin-3 protein), at least one additional binding site against a second antigen (i.e., different from trypsin-3 protein), and at least one additional binding site against a third antigen (i.e., different from the first and second antigens); and so on.

[0064] In some embodiments, the polypeptide is as described in WO2006064136. Specifically, the polypeptide may consist of: (i) a first fusion protein, wherein the CL constant domain of the antibody is fused via its N-terminus to the C-terminus of the single-domain antibody of the present invention (i.e., a single-domain antibody against trypsin-3 protein); and (ii) a second fusion protein, wherein the CH1 constant domain of the antibody is fused via its N-terminus to the C-terminus of a single-domain antibody against an antigen different from trypsin-3 protein. In another specific embodiment, the polypeptide consists of: a first fusion protein, wherein the CH1 constant domain of the antibody is fused via its N-terminus to the C-terminus of a single-domain antibody against an antigen different from trypsin-3 protein; and a second fusion protein, wherein the CL constant domain of the antibody is fused via its N-terminus to the C-terminus of the single-domain antibody of the present invention (i.e., against trypsin-3 protein).

[0065] In some embodiments, the polypeptide is a bicomplementary polypeptide. As used herein, the term "bicomplementary" polypeptide refers to a polypeptide comprising a single-domain antibody and a second single-domain antibody as defined herein, wherein these two single-domain antibodies are capable of binding to two different epitopes on the same antigen (e.g., trypsin-3 protein), epitopes that are typically not simultaneously bound by a single-specific immunoglobulin (e.g., a conventional antibody or a single-domain antibody). The bicomplementary polypeptide according to the invention consists of single-domain antibodies with different epitope specificities and does not contain mutually complementary pairs of variable domains that bind the same epitope. Therefore, they do not compete with each other for binding to trypsin-3 protein.

[0066] In some embodiments, the two single-domain antibodies of the dual complementary site polypeptide of the present invention can be directly linked to each other (i.e., without using a adapter) or linked to each other through a adapter.

[0067] The linker is typically a linker peptide, and according to the invention, its selection should enable each of the two single-domain antibodies to bind to at least two distinct epitopes on the trypsin-3 protein. A suitable linker depends particularly on the epitopes, specifically on the distance between the epitopes on the trypsin-3 protein to which the single-domain antibodies bind. Suitable linkers can be readily determined by those skilled in the art based on this disclosure, and if necessary, can be determined through limited conventional experiments. Furthermore, two single-domain antibodies binding to trypsin-3 protein can also be interconnected by a third single-domain antibody (the two single-domain antibodies can be directly linked to this third single-domain antibody, or linked through a suitable linker). Such a third single-domain antibody can, for example, be a single-domain antibody providing an extended half-life. For example, another single-domain antibody could be a single-domain antibody capable of binding (human) serum proteins such as (human) serum albumin or (human) transferrin, as will be further described herein. In some embodiments, two or more single-domain antibodies binding to trypsin-3 protein are linked in tandem (either directly or through a suitable linker); and the aforementioned third single-domain antibody providing an extended half-life can be directly or through a linker linked to any one of the two or more single-domain antibodies. Suitable linkers are described herein in conjunction with specific peptides of the invention and may—for example, but not limited to—comprising an amino acid sequence, preferably having 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 rather chosen for reasons such as facilitating the biopharmaceutical production of such peptides. The linker sequence may be a naturally occurring or non-natural sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in subjects administering the antitrypsin-3 protein peptide of the invention. A useful set of linker sequences are those derived from the hinge region of heavy chain antibodies as described in WO96 / 34103 and WO94 / 04678. Other examples are polyalanine linker sequences, such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly / Ser linkers of varying lengths, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0068] In some embodiments, the polypeptide comprises at least one single-domain antibody according to the invention.

[0069] In some embodiments, the polypeptide comprises at least two single-domain antibodies according to the invention.

[0070] In some embodiments, the polypeptide comprises two single-domain antibodies according to the invention.

[0071] In some embodiments, the polypeptide of the present invention comprises at least one single-domain antibody, which contains (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0072] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies, which contain (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0073] In some embodiments, the polypeptide of the present invention comprises 2, 3, 4 or 5 single-domain antibodies, which contain (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 20.

[0074] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% identity with the sequence shown in SEQ ID NO: 4.

[0075] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies that have at least 70% identity with the variable heavy chain (VH) sequences shown below: SEQ ID NO: 1 (“NT3-7”), SEQ ID NO: 5 (“NT3-12”), SEQ ID NO: 9 (“NT3-1”), or SEQ ID NO: 13 (“NT3-3”), or SEQ ID NO: 17 (“NT3-16”).

[0076] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having the following variable heavy chain (VH) sequences: SEQ ID NO: 1 (“NT3-7”), and / or SEQ ID NO: 5 (“NT3-12”), and / or SEQ ID NO: 9 (“NT3-1”), and / or SEQ ID NO: 13 (“NT3-3”), and / or SEQ ID NO: 17 (“NT3-16”).

[0077] In some embodiments, the polypeptide of the present invention comprises 2, 3, 4 or 5 single-domain antibodies having the following variable heavy chain (VH) sequences: SEQ ID NO: 1 (“NT3-7”), and / or SEQ ID NO: 5 (“NT3-12”), and / or SEQ ID NO: 9 (“NT3-1”), and / or SEQ ID NO: 13 (“NT3-3”), and / or SEQ ID NO: 17 (“NT3-16”).

[0078] According to the present invention, the polypeptide of the present invention as described above retains the biological activity of a single-domain antibody comprising or composed of the following variable heavy chain (VH) sequences: SEQ ID NO: 1 (“NT3-7”), or SEQ ID NO: 5 (“NT3-12”), or SEQ ID NO: 9 (“NT3-1”), or SEQ ID NO: 13 (“NT3-3”), or SEQ ID NO: 17 (“NT3-16”).

[0079] Therefore, in some embodiments, the polypeptide of the present invention comprises at least one trypsin-3 neutralizing single-domain antibody (which inhibits the protease activity of trypsin-3), such as NT3-7 or NT3-12.

[0080] In some embodiments, the polypeptide of the present invention comprises a sequence of a variable heavy chain (VH) as shown in SEQ ID NO: 1 (“NT3-7”) and / or a sequence of a variable heavy chain (VH) as shown in SEQ ID NO: 5 (“NT3-12”).

[0081] In some embodiments, the polypeptide of the present invention comprises (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; and / or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8.

[0082] In some embodiments, the polypeptide of the present invention binds to recombinant or human trypsin-3 protein, but does not bind to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms.

[0083] In a specific embodiment of a trypsin-3 single-domain antibody with dual complementary sites (any single-domain antibody of the present invention, particularly single-domain antibodies with “NT3-7” and “NT3-12”), and as demonstrated experimentally, computer simulation analysis based on a trypsin-3 D structural model was performed to optimize the linker, thereby promoting the interaction of the two single-domain antibodies on the trypsin-3 target. A linker with a potential length of 70.5 Å was designed to impart sufficient flexibility to the biologically active dual complementary site molecule.

[0084] Therefore, in this specific embodiment, the linker peptide length between the single-domain antibody "NT3-7" (whose variable heavy chain (VH) sequence is shown in SEQ ID NO: 1) and the single-domain antibody "NT3-12" (whose variable heavy chain (VH) sequence is shown in SEQ ID NO: 5) is 70.5 Å ± 5 Å.

[0085] The inventors further designed the linker sequence to avoid using amino acids that are known or predicted to be protease-sensitive sites.

[0086] In one specific embodiment, the linker between the single-domain antibody of the present invention, particularly “NT3-7” (the sequence of the variable heavy chain (VH) as shown in SEQ ID NO: 1) and “NT3-12” (the sequence of the variable heavy chain (VH) as shown in SEQ ID NO: 5), has the following sequence: GGGGSGGGGSAGSAAGSGEGGGGSGGGGSGGG (SEQ ID NO: 21).

[0087] In another specific embodiment, the linker between the single-domain antibody of the present invention, in particular “NT3-7” (the sequence of the variable heavy chain (VH) as shown in SEQ ID NO: 1) and “NT3-12” (the sequence of the variable heavy chain (VH) as shown in SEQ ID NO: 5), has the following sequence: GGGSGGGSAGSAAGSGEGGGSGGG (SEQ ID NO: 30).

[0088] The present invention provides a method for producing antibodies and immunoconjugates. The methods for obtaining such antibodies are well known in the art.

[0089] Camel Ig can be genetically engineered to produce small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "single-domain antibodies" or "VHHs". See U.S. Patent 5,759,808, issued June 2, 1998; also see (Cortez-Retamozo et al., 2002; Dumoulin et al., 2003; Lauwereys et al., 1998; Pleschberger et al., 2003; Stijlemans et al., 2004). Engineered libraries of camel antibodies and antibody fragments are commercially available from, for example, Ablynx, Ghent, Belgium / Sanofi, Gentilly, France. In some embodiments herein, single-chain camel antibodies or single-domain antibodies are naturally produced in camels, i.e., produced by immunizing camels with trypsin-3 protein or a peptide fragment thereof, using the techniques described herein for other antibodies. Subsequently, trypsin-3 protein-binding camelid single-domain antibodies (VHHs) were engineered from camelid single-chain antibodies. For example, using trypsin-3 protein as a target, a panning procedure was used to select from phage libraries displaying appropriately mutagenized camelid single-domain antibody (VHH) proteins. Alternatively, trypsin-3 protein could be used as a target, and VHHs could be selected from native phage libraries (unimmunized) using a panning procedure (see Moutel et al., 2016).

[0090] The single-domain antibodies of the present invention can be conjugated with detectable markers to form immunoconjugates. Suitable detectable markers include, for example, radioisotopes, fluorescent markers, chemiluminescent markers, enzyme markers, bioluminescent markers, or colloidal gold. Methods for preparing and detecting such immunoconjugates with detectable markers are well known to those skilled in the art and will be described in more detail below.

[0091] The detectable marker can be a radioactive isotope that can be detected by autoradiography. Isotopes particularly suitable for the purposes of this invention include 3H, 125I, 131I, 35S, and 14C.

[0092] Immunoconjugates can also be labeled with fluorescent compounds. The presence of fluorescently labeled antibodies is determined by exposing the immunoconjugate to light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescently labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein.

[0093] Alternatively, immunoconjugates can be detectably labeled by conjugating antibodies to chemiluminescent compounds. The presence of chemiluminescently labeled immunoconjugates can be determined by detecting the luminescent signal generated during the chemical reaction. Examples of chemiluminescently labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0094] Similarly, bioluminescent compounds can be used to label the immunoconjugates of this invention. Bioluminescence is a type of chemiluminescence present in biological systems, in which catalytic proteins enhance the luminescence efficiency of a chemical reaction. The presence of bioluminescent proteins is determined by detecting the presence of luminescence. Bioluminescent compounds that can be used for labeling include luciferin, luciferase, and jellyfish luminescent proteins.

[0095] Alternatively, immunoconjugates can be detectably labeled by attaching antibodies to enzymes. When an enzyme conjugate is co-incubated with a suitable substrate, the enzyme moiety reacts with the substrate to generate a chemical group that can be detected, for example, by spectrophotometry, fluorescence, or visual inspection. Examples of enzymes that can be used for multispecific immunoconjugate labeling include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0096] The antibodies of this invention can be labeled with metallic chemical elements such as lanthanides. Compared with other labels, lanthanides have several advantages: they are stable isotopes, numerous in variety, providing more than 100 different labels; they are relatively stable, their signals are easily identifiable in mass spectrometry detection, and signals from different detection channels are easily distinguishable. Lanthanide labeling also has a wide detection dynamic range, high detection sensitivity, and is insensitive to light and time, thus offering flexibility, strong stability, and applicability to various environments. Lanthanides are fifteen metallic chemical elements with atomic numbers 57 to 71, also known as rare earth elements. Lanthanides can be detected using CyTOF technology. CyTOF is inductively coupled plasma time-of-flight mass spectrometry (ICP-MS). CyTOF instruments can analyze up to 1000 cells per second, analyzing a number of parameters comparable to the number of available stable isotope labels.

[0097] Those skilled in the art will recognize other suitable markers that can be used in this invention. The binding of the labeled portion to the single-domain antibody can be accomplished using standard techniques known in the art.

[0098] Furthermore, the use of monoclonal antibodies conjugated with avidin, streptavidin, and biotin can enhance the convenience and versatility of immunochemical assays.

[0099] The single-domain antibodies of the present invention can be produced by any technology known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technology, and can be used alone or in combination.

[0100] Once the amino acid sequence of the desired sequence is known, those skilled in the art can readily prepare the antibody using standard techniques for producing peptides. For example, it can be synthesized using known solid-phase methods, preferably using commercially available peptide synthesis equipment (e.g., equipment manufactured by Applied Biosystems, Foster City, California) and operated according to the manufacturer's instructions. Alternatively, the antibody of the present invention can be synthesized using recombinant DNA techniques known in the art. For example, the DNA sequence encoding the antibody is inserted into an expression vector, which is then introduced into a suitable eukaryotic or prokaryotic host cell; after the host cell expresses the desired antibody, the antibody can be isolated from it using conventional known techniques.

[0101] Therefore, another object of the present invention relates to a nucleic acid sequence encoding the single-domain antibody of the present invention.

[0102] In one specific embodiment, the present invention relates to a nucleic acid sequence encoding the VH domain of an antibody of the present invention (e.g., a single-domain antibody NT3-X). Examples of nucleic acid sequences encoding the VH domain of NT3 in plasmids are described in SEQ ID NO: 24 (NT3-1), SEQ ID NO: 25 (NT3-3), SEQ ID NO: 26 (NT3-7), SEQ ID NO: 27 (NT3-12), and SEQ ID NO: 28 (NT3-16).

[0103] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, granule, episome, artificial chromosome, bacteriophage, or viral vector.

[0104] The terms “vector,” “cloning vector,” and “expression vector” refer to vectors that can introduce DNA or RNA sequences (such as foreign genes) into host cells, thereby transforming the host and promoting the expression of the introduced sequence (such as transcription and translation).

[0105] Therefore, another object of the present invention relates to vectors comprising the nucleic acids of the present invention. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to induce or direct the expression of said antibodies upon administration to a subject. Examples of promoters and enhancers for animal cell expression vectors include early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney mouse leukemia virus, promoters and enhancers of immunoglobulin H chains, etc. Examples of plasmids include replication plasmids containing the origin of replication, or integration plasmids, such as pUC, pcDNA, pBR, etc. Examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be produced using techniques known in the art, such as by transfection of packaging cells or by transient transfection using helper plasmids or viruses.

[0106] Another object of the present invention relates to a host cell that has been transfected, infected or transformed with the nucleic acid and / or vector of the present invention and expresses the single-domain antibody of the present invention.

[0107] Therefore, such recombinant host cells can be used to generate the antibodies of the present invention.

[0108] The term "transformation" refers to the introduction of a "foreign" (i.e., foreign or extracellular) gene, DNA, or RNA sequence into a host cell, thereby enabling the host cell to express the introduced gene or sequence to produce the desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed."

[0109] The nucleic acids of this invention can be used to produce the antibodies of this invention in a suitable expression system. The term "expression system" refers to a host cell and compatible vector under suitable conditions, for example, for expressing a protein encoded by exogenous DNA carried by a vector and introduced into 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. Examples of other host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include *E. coli* (…). E. coli Kluyveromycin ( Kluyveromyces ) or yeast ( ) Saccharomyces Mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (such as cells derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub & Chasin, 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc.

[0110] The present invention also relates to a method for producing the single-domain antibody of the present invention, the method comprising the following steps: (i) introducing the recombinant nucleic acid or vector as described above into competent host cells in vitro or in vitro, (ii) culturing the obtained recombinant host cells in vitro or in vitro, and (iii) recovering the expressed antibody.

[0111] The detection and diagnostic method of the present invention: The single-domain antibodies and immunoconjugates of this invention can be used to detect human trypsin-3 protein and / or assess the content of this protein in biological samples, particularly culture media, whole blood, serum, plasma, or any tissue samples. Therefore, they can be used to diagnose all diseases associated with abnormal trypsin-3 activity.

[0112] Therefore, the detection method of the present invention can be used for in vitro diagnosis of pathological diseases related to trypsin-3.

[0113] The term "trypsin-3-related pathological diseases" has its general meaning in this field, referring to diseases characterized by abnormal trypsin-3 activity. Trypsin-3-related pathological diseases include intestinal diseases related to intestinal permeability, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis, and also include cancers, particularly cancers related to trypsin-3.

[0114] The terms "cancer" and "tumor" refer to or describe a pathological state in mammals typically characterized by unregulated cell growth. More precisely, in the context of this invention, diseases that express / secrete trypsin-3 (i.e., tumors) are most likely to be detected or responded to by the trypsin-3 single-domain antibody of this invention. In particular, the cancer may be associated with solid tumors or lymphomas / leukemias (derived from hematopoietic cells). Examples of cancers associated with solid tumor formation include: breast cancer, uterine / cervical cancer, esophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, gastric cancer, mesenchymal tumors (i.e., fibrosarcoma and rhabdomyosarcoma), central and peripheral nervous system tumors (including astrocytomas, neuroblastomas, gliomas, glioblastomas), and / or thyroid cancer.

[0115] In one specific implementation, solid tumors are cancers associated with trypsin-3.

[0116] The term "cancer associated with trypsin-3" refers to tumors that express and secrete trypsin-3. Examples of cancers 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, ApplImmunohistochem Mol Morpho 2021, Mar 22), lung adenocarcinoma (MA, scientific reports (2019)9:1844), pancreatic cancer (Jiang G. Gut 2010;59:1535e1544. Doi:10.1136 / gut.2009.200105), ovarian cancer (MA R., Gynecologic Oncology 137 (2015) 546–552), and breast cancer (Qian L., Oncotarget, 2017, Vol. 8, (No. 13), pp: 21444-21453), gastric cancer (Fei Wang MS, J SurgOncol. 2019;119:1108-1121.), endometrial cancer (Aboulouard Cell Reports Medicine 2,100318 June 15, 2021 a 2021), large B-cell lymphoma (Hindawi Disease Markers Volume2022, Article ID 1254790, 9 pages), esophageal cancer (Han S, Lee CW, Trevino JG, HughesSJ, Sarosi GA Jr (2013), PloS ONE 8(10): e76667. Doi:10.1371 / journal.pone.0076667).

[0117] Preferably, the solid tumors and cancers associated with trypsin-3 are selected from the group consisting of pancreatic cancer and prostate cancer.

[0118] More preferably, pancreatic cancer is pancreatic ductal adenocarcinoma.

[0119] One object of the present invention is to provide a method for detecting and / or assessing the content of human trypsin-3 protein in a biological sample, wherein the method comprises: contacting the sample with the antibody or immunoconjugate under conditions that allow the formation of an immune complex between the human trypsin-3 protein and the antibody or immunoconjugate of the present invention, and detecting or measuring the formed immune complex.

[0120] The resulting immune complexes can be detected or measured using a variety of standard techniques, including, but not limited to, enzyme-linked immunosorbent assay (ELISA) or other solid-phase immunoassays, radioimmunoassays, electrophoresis, immunofluorescence, or Western blotting.

[0121] Another object of the present invention is to provide a method for diagnosing trypsin-3-related pathological diseases, wherein the method comprises: as described above, assessing the content of human trypsin-3 protein in a biological sample of a subject to be tested, and comparing the measured content with the control value of trypsin-3 in a normal subject.

[0122] Finally, the present invention also provides a kit comprising at least one single-domain antibody of the present invention. The kit of the present invention may comprise a single-domain antibody conjugated to a solid support (e.g., a tissue culture plate or microbeads, such as agarose microbeads). The provided kit may comprise antibodies for the in vitro detection and quantification of trypsin-3 protein, for example, for ELISA or Western blotting. Such single-domain antibodies for detection may be labeled, for example, fluorescently labeled or radiolabeled.

[0123] The treatment method of the present invention: According to the present invention, any of the above-described antitrypsin-3 (particularly neutralizing) single-domain antibodies; cross-competitive antitrypsin-3 (particularly neutralizing) single-domain antibodies; polypeptides comprising at least one antitrypsin-3 (particularly neutralizing) single-domain antibody; nucleic acid sequences encoding antitrypsin-3 (particularly neutralizing) single-domain antibodies; and vectors comprising nucleic acids encoding the previously described antitrypsin-3 (particularly neutralizing) single-domain antibodies are all suitable for therapeutic purposes. Another object of the present invention relates to a pharmaceutical composition comprising the antitrypsin-3 neutralizing single-domain antibody of the present invention, or the cross-competitive neutralizing single-domain antibody of the present invention, or a polypeptide comprising at least one neutralizing single-domain antibody of the present invention, or a nucleic acid sequence encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention, or a vector comprising nucleic acids encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention.

[0124] Therefore, in some embodiments, the pharmaceutical compositions of the present invention comprise at least one trypsin-3 neutralizing single-domain antibody (which inhibits the proteolytic activity of trypsin-3), such as NT3-7 or NT3-12 or the cross-competitive neutralizing single-domain antibody of the present invention. Alternatively, they may comprise a polypeptide comprising at least one neutralizing single-domain antibody of the present invention (e.g., NT3-7 or NT3-12), or a nucleic acid sequence encoding the anti-trypsin-3 neutralizing single-domain antibody of the present invention (e.g., NT3-7 or NT3-12), or a vector comprising the nucleic acid encoding the anti-trypsin-3 neutralizing single-domain antibody of the present invention (e.g., NT3-7 or NT3-12).

[0125] Another object of the present invention relates to a pharmaceutical composition comprising the antitrypsin-3 neutralizing single-domain antibody of the present invention or the cross-competitive antitrypsin-3 neutralizing single-domain antibody of the present invention, or a polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody of the present invention or a nucleic acid sequence encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention or a carrier comprising a nucleic acid encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention, said pharmaceutical composition for therapeutic purposes.

[0126] Another object of the present invention relates to a pharmaceutical composition comprising the antitrypsin-3 neutralizing single-domain antibody of the present invention, the cross-competitive antitrypsin-3 neutralizing single-domain antibody of the present invention, or a polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody of the present invention, or a nucleic acid sequence encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention, or a carrier comprising nucleic acid encoding the antitrypsin-3 neutralizing single-domain antibody of the present invention, said pharmaceutical composition for treating trypsin-3-related pathological diseases.

[0127] As previously stated, the term "trypsin-3-related pathology" has its general meaning in the art and refers to diseases characterized by abnormal trypsin-3 activity. Trypsin-3-related pathology includes intestinal diseases related to intestinal permeability, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis, and also includes cancers, especially cancers related to trypsin-3.

[0128] Another object of the present invention relates to a method for treating trypsin-3-related pathology, the method comprising administering to a subject in need a therapeutically effective amount of the present invention's antitrypsin-3 neutralizing single-domain antibody, the present invention's cross-competitive antitrypsin-3 neutralizing single-domain antibody, or a polypeptide comprising at least one of the present invention's antitrypsin-3 neutralizing single-domain antibodies, or a nucleic acid sequence encoding the present invention's antitrypsin-3 neutralizing single-domain antibody, or a vector comprising a nucleic acid encoding the present invention's antitrypsin-3 neutralizing single-domain antibody.

[0129] In some embodiments, a method of treating a subject with trypsin-3-related pathology is carried out by administering an antitrypsin-3 neutralizing single-domain antibody, a cross-competitive antitrypsin-3 neutralizing single-domain antibody, or a polypeptide comprising at least one (i.e., 1, 2, 3, 4, 5, or more) antitrypsin-3 neutralizing single-domain antibody, wherein the neutralizing single-domain antibody comprises an element that binds to human trypsin-3, and wherein the antitrypsin-3 neutralizing single-domain antibody: (i) Not bound to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms; (ii) Further demonstrated the ability to inhibit active human trypsin-3 (neutralizing antibody); and / or (iii) The KD binding to human active trypsin-3 protein is 200 nM or less, 100 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, or 0.05 nM or less.

[0130] In some embodiments, the antitrypsin-3 neutralizing single-domain antibody, the cross-competitive antitrypsin-3 neutralizing single-domain antibody, or the polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody includes: (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; and / or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8.

[0131] In some embodiments, the polypeptide comprises at least one single-domain antibody having at least 70% identity with the sequence of a variable heavy chain (VH) as shown in SEQ ID NO: 1 (“NT3-7”) and / or SEQ ID NO: 5 (NT3-12).

[0132] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having sequences of variable heavy chains (VH) as shown in SEQ ID NO: 1 (“NT3-7”) and / or SEQ ID NO: 5 (NT3-12).

[0133] The "therapeutic effective dose" of the single-domain antibody of the present invention refers to a sufficient amount to treat the aforementioned trypsin-3-related pathology and to have a reasonable benefit / risk ratio suitable for any medical treatment. However, it should be understood that the total daily dosage of the antibody and composition of the present invention will be determined by the attending physician within the bounds of reasonable medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific antibody used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific antibody used; the duration of treatment; drugs used in combination with or concurrently with the specific antibody used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art that the starting dose of the compound is below the level required to achieve the desired therapeutic effect, and the dose is gradually increased until the desired effect is achieved.

[0134] When administered, the single-domain antibody or fragment thereof of the present invention is formulated into a pharmaceutical composition. Pharmaceutical compositions comprising the antibody or fragment thereof of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, wherein the therapeutic molecule is mixed with a pharmaceutically acceptable carrier. A composition is referred to as a "pharmaceutically acceptable carrier" if its administration is tolerated by the treated patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc. The form, route of administration, dosage, and regimen of the pharmaceutical composition naturally depend on factors such as the disease to be treated, the severity of the disease, the patient's age, weight, and sex. The pharmaceutical compositions of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. To prepare the pharmaceutical composition, an effective amount of antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The pharmaceutical forms include sterile aqueous solutions or dispersions; preparations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. The composition must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The single-domain antibodies of the present invention can be formulated into compositions in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino group of a protein) and those formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate flowability can be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. The absorption time of injectable compositions can be prolonged by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0135] The single-domain antibody of the present invention can be formulated into a therapeutic mixture containing about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 mg, or even about 10 mg per dose. Multiple doses can also be administered.

[0136] Uses of food-grade bacteria Using food-grade bacteria (or probiotics) to deliver the antitrypsin-3 single-domain antibody of the present invention or a polypeptide containing at least one antitrypsin-3 single antibody, especially the antitrypsin-3 neutralizing single-domain antibody of the present invention or a polypeptide containing at least one antitrypsin-3 neutralizing single-domain antibody of the present invention, can safely and efficiently target the above compounds locally in the intestine, enabling the diagnosis and treatment of intestinal-related diseases.

[0137] Therefore, another object of the present invention relates to a recombinant food-grade bacterium comprising a gene or nucleic acid sequence encoding an antitrypsin-3 single-domain antibody of the present invention or a polypeptide comprising at least one antitrypsin-3 single-domain antibody of the present invention (e.g., NT3-X sdAb), for (1) detecting and / or diagnosing "trypsin-3-related pathology" (e.g., intestinal diseases related to intestinal permeability, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis); and / or (2) treating patients with intestinal diseases related to intestinal permeability (IBD, IBS, celiac disease, or pouchitis), wherein the recombinant food-grade bacterium is locally administered into the intestine of the patient to be treated.

[0138] Therefore, in order to be applied directly to the intestine (gut), the recombinant food-grade bacteria of the present invention are preferably administered orally (including oral and sublingual administration), rectally or locally (colonic administration) to patients with IBD or IBS.

[0139] Another aspect of the present invention relates to therapeutic compositions comprising recombinant food-grade bacteria as defined above.

[0140] Another aspect of the invention relates to an antitrypsin-3 neutralizing single-domain antibody of the invention or a polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody of the invention for treating patients with intestinal diseases associated with intestinal permeability (e.g., irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis), wherein food-grade bacteria comprising a gene or nucleic acid sequence encoding the antitrypsin-3 single-domain antibody of the invention or a polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody (e.g., NT3-7 and / or NT3-12 single-domain antibody) are used to directly deliver the neutralizing single-domain antibody or the polypeptide comprising at least one antitrypsin-3 neutralizing single-domain antibody to the intestine.

[0141] As used herein, the term "food-grade bacteria" refers to bacteria widely used in fermented foods that are completely safe and are recognized as GRAS (Generally Recognized As Safe) in the United States and QPS (Qualified Presumption of Safety) in the European Union. These bacteria can be safely used in functional foods or food additives and can be claimed to help maintain health, promote wellness, or prevent disease.

[0142] As used in this article, the term "probiotics" refers to bacteria that, when ingested in sufficient quantities, have a beneficial effect on human health. Currently, probiotics are widely used as food additives due to their health-promoting effects. Most probiotics are lactic acid bacteria (LAB), with strains of the genera *Lactobacillus* and *Bifidobacterium* being the most widely used types.

[0143] In a preferred embodiment, the food-grade bacterial strain of the present invention is *Lactococcus lactis* (Lactococcus lactis). Lactococcus lactis ) strain, Lactobacillus casei ( Lactobacillus casei ) strain, Lactococcus lactis htrA strain (Poquet et al., Molecular Microbiology (2000) 35(5), 1042–1051), Lactobacillus plantarum ( Lactobacillus plantarum ) strain, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) strain, Bifidobacterium longum ( Bifidobacterium longum ) strain or Escherichia coli Nissle 1917 strain (Chen H et al., MaterToday Bio. 2023 Feb; 18: 100543).

[0144] In a preferred embodiment, the food-grade bacterial strain used in this invention is Escherichia coli Nissle1917.

[0145] The CDR3-derived peptide of the antitrypsin-3 neutralizing antibody of the present invention: The inventors have also demonstrated that the CDR3 of the trypsin-3 neutralizing antibodies (“NT3-7” and “NT3-12” sdAbs) of the present invention has the potential to inhibit the protease activity of trypsin-3 and thus serve as a specific trypsin-3 protease inhibitor (see below, “Bioactivity of the antibodies of the present invention as determined by protease activity assay”). In fact, previous studies have shown that peptides derived from the CDR3 region of neutralizing antibodies can also be used alone to neutralize original therapeutic targets in the field of infection, as shown in HIV treatment (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).

[0146] Therefore, the present invention also covers a polypeptide comprising: (a) CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; and / or (b) CDR3 having NT3-12 sdAb with the sequence shown in SEQ ID NO: 8.

[0147] Another object of the present invention relates to a therapeutic polypeptide comprising or composed of the following: (a) CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; and / or (b) CDR3 having NT3-12 sdAb with the sequence shown in SEQ ID NO: 8.

[0148] Another object of the present invention relates to a polypeptide comprising or composed of the following: (a) CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; and / or (b) CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8. It is used to treat trypsin-3-related pathologies.

[0149] The present invention will be further illustrated by the following figures and embodiments. However, these embodiments and figures should not be construed as limiting the scope of the invention in any way. Attached Figure Description

[0150] Figure 1. Inhibition of trypsin-3 by NT3-7 and NT3-12.

[0151] To quantify the ability of NT3-7 and NT3-12 to control trypsin-3 activity, trypsin-3 activity inhibition curves were plotted as the concentration of the single-domain antibody increased. The half-maximal inhibitory concentration (IC50) is expressed as a semi-logarithmic figure (a, b).

[0152] To determine the inhibition mode, initial reaction rate curves were plotted at varying concentrations of the peptide substrate Z-GPR-pNA and NT3; the lines fitted by multiple regression were superimposed on the data points (c, d). All studies were conducted at a final trypsin-3 concentration of 0.5 nM and substrate concentrations ranging from 25 to 250 µM (as shown on the x-axis).

[0153] All figures represent the results of three independent experiments. The epigenetic inhibition constant was calculated using Lineweaver-Burk plots generated from these data (d).

[0154] Figure 2The inhibitory specificity of the screened antitrypsin-3 single-domain antibodies Before adding the substrate, the protease was pre-incubated with the single-domain antibody at an I / E ratio of 1000 for 15 minutes. NR single-domain antibody: An unrelated single-domain antibody served as a negative control.

[0155] Figure 3. Precautions for developing dual epitopes.

[0156] The table describes different possible combinations between trypsin-3 inhibitors and other sdAbs exhibiting high affinity for trypsin-3 (a). Potential interactions between two single-domain antibodies on trypsin-3 were detected by a sandwich ELISA assay; NT3.7 or NT3.12 were coated into wells (b). Computer simulation analysis based on the 3D structure of trypsin-3 was used to determine the longest distance between two distal epitopes on the protease.

[0157] Figure 4. Characterization of dual epitopes.

[0158] Compared to single-epitope sdAbs, dual-epitope single-domain antibodies exhibit inhibitory properties against trypsin-3 activity. The curves represent the percentage of protease activity (a) obtained based on the concentration (I, nM) of the single-domain antibody added to the reaction mixture.

[0159] Single-cycle kinetic surface plasmon resonance measurements of trypsin-3 immobilized on a Ni-NTA chip (captured via nickel affinity with a 6×His tag) (b). Thick lines correspond to the raw measurements, and fitted curves are shown as thin lines. The analyte was recombinant NT3-7 / 12, injected at increasing concentrations (1.5, 3.12, 6.25, 12.25, and 25 nM; arrows indicate).

[0160] Figure 5. Effect of NT3 single-domain antibody on the malignant growth of PC3 prostate cancer cells. PC3 cells were treated for 24 hours with NT3-7 / 12, NT3-7 / 7 (as a negative control for trypsin-3 inhibition), or buffer (-) alone, ranging from 100 to 1000 nM. Cell roundness was measured after fixation and Phalloidin staining to delineate the cell surface. An index of 1 was given for round objects, and an index of less than 1 was given for objects deviating from a round shape. Results are the mean and SEM of at least 200 characteristic cells. (a) Two-way ANOVA was used, with post-hoc tests corrected using Tukey, p-values ​​compared under specified conditions: **p<0.002, ***p<0.0002, ****p<0.0001. To assess the effect of the inhibitor NT3 on the migration ability of PC3 cells, cells were cultured in different compartments for 24 hours and then treated for 14 hours with NT3-7 / 12, NT3-7 / 7 (as a negative control for trypsin-3 inhibition), or buffer (-) alone, ranging from 25 to 1000 nM. Images were taken immediately after wound incision (0 hours), hourly for 14 hours, to monitor cell migration into the gaps. The gap closure rate was calculated and plotted in Figure (b). One-way ANOVA was used, with post-hoc tests corrected for Tukey. p-values ​​for comparison with the untreated condition: ** p < 0.002, *** p < 0.0002. Detailed Implementation

[0161] Example 1: Selection of VHHs targeting trypsin-3 Materials and methods plasmid Using the previously described pAOT7 expression vector, any humanized synthetic single-domain antibody (Nb) from the NALI-H1 library can be inserted between the NcoI and NotI restriction sites (Moutel, S., et al., NaLi-H1: Auniversal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife, 2016.). This vector can produce recombinant proteins in the cytoplasm of *E. coli*. The 6His-Myc-6His tag located downstream of the NotI site encodes the following translational sequence: HHHH HH GAAEQKLISEEDLNGGSPVGR HHHHHH* (SEQ ID NO: 22), thereby producing the pAOT7-Nb-6His-myc-6His form. The 6His-myc-6His tag downstream of the NotI site was replaced with a synthetic Flag-Ctag DNA fragment encoding the following translated sequence: AAAGGGSGGDYKDDDDKGY QD YEPEA * (SEQ ID NO: 23), resulting in the pAOT7-NB-Flag-ctag form, as previously described (Keller, Tardy, Analytical Chemistry 2021).

[0162] The pCMV-IL2ss-Trypsin-2x-Strep-tag and pCMV-IL2ss-Trypsin-CBD-tag plasmid expression vectors contain the IL2 signaling sequence (IL2ss) that facilitates protein secretion. Ampicillin resistance screening was performed before amplification of the pCMV plasmids.

[0163] Trypsin-1 and trypsin-2 inactive protease mutants were obtained through gene synthesis. These were codon-optimized cDNA sequences of human pre-trypsin-3 and S195A mutant human trypsin-1, -2, and -3, located downstream of the interleukin-2 secretion signal sequence, enabling efficient and uniform secretory expression of the proteases. Using AflIII and NotI restriction sites, the constructs were subcloned into two different pCMV plasmids derived from pcDNA3.1 (Thermo Fisher). The modified plasmids expressed proteins fused with a C-terminal purification tag, which could be either a 2xStrep-tag® (similar to the Twin-Strep-tag® from IBA-Lifesciences) or a chitin-binding domain.

[0164] A dual-epitope single-domain antibody (Genecust, Boynes, France) consisting of two single-domain antibody sequences linked by a flexible linker sequence (GGGGSGGGGSAGSAAGSGEGGGGSGGGGSGG SEQ ID NO: 21) was chemically synthesized using artificial gene synthesis. This antibody was then subcloned into an empty plasmid digested with pAOT7-NcoI-NotI-FlagCTAG, resulting in the constructed pAOT7-hs2dAb1-linker-hs2dAb2-FlagCtag plasmid. For all pAOT7 plasmids expressed and purified, kanamycin resistance was determined using the BL21(DE3)pLysS strain (L1195, Promega).

[0165] Expression and purification of trypsin.

[0166] HEK293F cells were cultured in serum-free Freestyle™ 293 expression medium (#12338018, Thermofisher) without any antibiotics in a humidified incubator at 37°C with shaking at 125 rpm. This medium was used for cell growth, maintenance, and transfection. Cells were passaged when the cell density reached 1×10^6 to 3×10^6 viable cells, and viability was determined using trypan blue. HEK293F cells were transfected using Freestyle MAX transfection reagent (16447100, Thermofisher) according to the supplier's instructions.

[0167] Attenuated phage display panning technique for isolating trypsin-3 specific single-domain antibodies This study utilized the humanized synthetic single-domain antibody library NaLi-H1. A subtractive panning protocol was designed to isolate Nb selectively targeting the mature conformation of trypsin-3. Immediately after cell lysis, chitin-binding domains (CBDs) from chitinase A1 or 2x-Strep-tag fusion proteins of trypsin-1, trypsin-2, trypsin-3 S195A mutants, and wild-type trypsin-3 were captured onto magnetic beads and then incubated with the library phages. During three rounds of phage display, Strep-Tactin®-coated magnetic beads or chitin-based magnetic beads were used alternately to capture the antigen. HEK293T cells (ATCC; Rockville, USA) were grown in Dulbecco modified Eagle medium (DMEM; Gibco BRL, Life Technology) supplemented with 10% heat-inactivated FBS (fetal bovine serum) (BioWittaker). HEK293T cells were passaged using PBS buffer to avoid the use of trypsin solution. As instructed by the supplier (Polyplus Transfection), cells were transiently transfected with plasmids expressing IL2ss-pro-Trypsins-2S or IL2ss-pro-Trypsins-CBD using JetPrime reagent. Four days after transfection, the supernatant was collected and co-incubated with magnetic MagStrep “type 3” XT (IBA-Lifesciences, 2-4090-002) or chitin-binding domain-coated magnetic beads (New England Biolabs, E8036) to capture and purify trypsin secreted into the supernatant. Before the first round of screening, the phage empty vector was adsorbed onto blank chitin magnetic beads (NEB) or MagStrep type 2 magnetic beads (IBA) to remove non-specifically bound phages. The second and third rounds of screening included a subtraction and removal step for mutant trypsin-1 and mutant trypsin-2. An appropriate amount of antigen-coated magnetic beads and a phage library (10¹¹ phages diluted in 1 mL TBSC + 0.01% Tween + 0.5% casein) were placed in a rotating shaker and incubated at 4°C for 1 hour. Phages bound to the antigen on MagStrep-coated or chitinous magnetic beads were recovered using a magnetic rack. The magnetic beads were washed with 0.1% TBS-Tween 10 times (round 1) and 25 times (round 2).

[0168] Expression and purification of single-domain antibodies Cytoplasmic expression of NB-6His-Myc-6His and NB-Flag-CTag was performed in BL21(DE3)pLysS *Escherichia coli* (#L1195, Promega) using the pAOT7 vector. Transformed bacterial cells were grown in 500 μL of initial medium containing 1% glucose and 3% ethanol (TB / kanamycin 35 μg / mL) and incubated at 37°C with shaking at 220 rpm for 2 h. Cells were then grown overnight at 37°C in 10 mL of TB / kanamycin 35 μg / mL medium containing 1% glucose and 3% ethanol. The pre-culture was then diluted in a 2 L flask containing 500 mL of the same medium containing 1% glucose and 3% ethanol. Cells were grown at 37°C until the OD600 reached 0.5–0.7, and then diluted with 500 mL of TB without glucose or ethanol to achieve a glucose concentration of 0.5%. When the OD600 reached 0.5–0.7, the cells were induced with IPTG (isopropyl-β-D-thiogalactopyranoside, 18824P, Sigma Aldrich) to a final concentration of 100 μM and regenerated at room temperature with stirring for 16 h. Cells were collected by centrifugation (30 min, 6000 g, 4 °C). After removing the supernatant, the precipitate was rapidly frozen in liquid nitrogen and stored at -80 °C before processing. The precipitate was incubated at 37 °C and resuspended in 10 mL of ice-cold lysis buffer (20 mM Tris, 150 mM NaCl pH 8, 1X lysozyme). They were kept on ice for 30 min, and the bacterial suspension was sonicated on ice for 15 pulses at 10-second intervals. The sonicated solution was centrifuged (30 min, 12000 g, 4 °C) to separate the precipitate from the supernatant containing soluble proteins. The supernatant was kept on ice and protein quantification was performed using a BCA assay (#23225, ThermoFisher). The protein was diluted to 1 mg / mL with an appropriate ice-cold buffer and filtered through a 0.45 μm filter membrane.

[0169] The supernatant containing Nb was then purified by affinity chromatography. Nb-6His-Myc-6His was purified using Ni-NTA-coated magnetic beads (Qiagen). The magnetic beads were washed with 2.5 mL of wash buffer (50 mM Na2HPO4 pH 8, 300 mM NaCl, 10 mM imidazole). The single-domain antibody was then eluted with elution buffer (50 mM Na2HPO4 pH 7, 500 mM NaCl, 300 mM imidazole) and dialyzed three times at 4°C with PBS 1X without CaCl2 or MgCl2 for 16 h each time, followed by two 2 h intervals. Purity was assessed by SDS-PAGE and subsequent Coomassie brilliant blue staining. After quantification by BCA method, glycerol was added to prepare a 20% glycerol solution. The aliquoted samples were rapidly frozen in liquid nitrogen and stored at -80°C.

[0170] NB-FlagCTag was purified using a 1 mL CS C-TAGXL MINICHROM (Thermofisher) column and CaptureSelect™ C-tag affinity matrix (ThermoFisher). The matrix was equilibrated with 2CV of 20 mM Tris, 150 mM NaCl pH8 buffer at a flow rate of 1 mg / mL. After injecting the clarified lysis buffer at a flow rate of 1 mg / mL, the column was washed with 10CV of 20 mM Tris, 150 mM NaCl pH8 buffer at a flow rate of 1 mg / mL. Finally, the C-tag single-domain antibody was eluted using a two-step isocratic elution process: the first step used 17% buffer containing 2 M MgCl2, 20 mM Tris, 150 mM NaCl pH8, and the second step used 100% of the above solution. The antibody was then dialyzed three times consecutively at 4°C with CaCl2- and MgCl2-free PBS1X, the first overnight and the next two 2-hour intervals. Residual MgCl2 has been shown to interfere with subsequent enzyme activity assays, therefore dialysis quality was strictly controlled. Purity was assessed by SDS-PAGE and subsequent Coomassie brilliant blue staining. After three rounds of dialysis and BCA quantification, glycerol was added to prepare a 20% glycerol solution. The aliquoted samples were rapidly frozen in liquid nitrogen and stored at -80°C.

[0171] Relative ELISA method for assessing antigen binding activity To identify single-domain antibodies for detecting captured trypsin, 20 to 50 nM of recombinant mature trypsin-2s was added to the wells of a Strep-Tactin-coated ELISA plate (IBA-LifeSciences®), and the plate was incubated overnight at 4°C. Then, it was blocked for 1 hour at room temperature with 0.05% TBSC-Tween (blocking buffer) containing 5% milk. Single-domain antibodies diluted in blocking buffer were added to the wells of the ELISA plate in duplicate and incubated at room temperature for 2 hours and 30 minutes. The interaction between trypsin and single-domain antibodies was visualized by incubating with anti-myc-HRP (18824P, QED Bioscience) for 1 hour at room temperature, followed by incubation with 100 µL of chromogenic substrate (Thermoscientific®, 1-step ultraTMB) for 5 to 30 minutes. The reaction was terminated with 50 μL of H₂SO₄ 1N, and the absorbance at 450 nm was measured using a FLUOstar OPTIMA microplate reader (BMG LABTECH, Ortenberg, Germany). After each step, wash the wells 1 to 3 times with washing buffer (TBSC-Tween20 0.05%). All steps were performed with shaking (40 rpm).

[0172] For capture or sandwich ELISA assays, the single-domain antibody with the 6His tag was diluted in TBS 1X-10mM CaCl2-0.05% Tween (TBSCT) to obtain a working concentration of 500 nM. This was then added to nickel-coated plates (15142, Life Technologies) according to the plate layout (100 μl per well) and incubated at room temperature with slow shaking for 1 hour. 100 µL of active trypsin-3-2S (200 nM) was added to each well. After three washes, except for the 2x-Streptag-coated control, 100 μL of Flag-labeled NT3 (NT3-1, NT3-3, NT3-7, NT3-12, and NT3-16) was added to each well, while the 2x-Streptag-coated control wells only received TBSCT-5% milk. After several washes, the His-tagged control was incubated with anti-myc antibody (18824P, QED Bioscience, 1:10000), the StrepTag-tagged control was incubated with Streptactin-HRP (2-1502-001, IBA, 1:500), and the assay wells were incubated with anti-Flag M2-HRP (A8592-2MG, Sigma Aldrich, 1:20,000) at room temperature with slow shaking for 1 hour. The reaction was terminated by adding 100 μL of chromogenic substrate (1-Step Ultra TMB, ThermoFisher) for 30 minutes, followed by adding 50 μL of 1N H2SO4 to visualize the ELISA results. Finally, the optical density (OD) was measured at 450 nm using a Varioskan Flash Reader (ThermoFisher).

[0173] Protease activity Trypsin-3 activity was determined using recombinant human active trypsin-3 (3714-SE-10, Bio-techne R&D), diluted in BOC-CHAPS-0.1% BSA buffer (100 mM Tris HCl, 1 mM CaCl2, 0.1% BSA, pH 8) to a final concentration of 0.5 nM. The solution was incubated at 37°C for 15 minutes with each single-domain antibody diluted in enzyme buffer. After incubation, N-p-toluenesulfonyl-GPR-amino-4-methylcoumarin hydrochloride (0.1 mM) substrate was added. Substrate degradation was calculated by measuring fluorescence changes (excitation wavelength 355 nm, emission wavelength 460 nm) using a Varioskan Flash microplate reader (Thermofisher) at 37°C for 20 minutes. The activities of thrombin (T6884, Sigma Aldrich) and kallikrein 5 (1108-SE-010, Biotechne-R&D) were as described previously. Prokallikrein 1 (2337-SE-010, Biotechne R&D) was activated by thermophilic protease (3097-ZN-020, Biotechne R&D) in activation buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5) at 37°C for 1 h. All activity assays were performed in assay buffer (50 mM CHES, 250 mM NaCl, pH 10).

[0174] IC50 was calculated using GraphPad Prism software by plotting the inhibition percentage against log[I]. To illustrate the effect of substrate KM on the inhibition constant, KI was calculated using Lineweaver Burk plots.

[0175] Affinity Measurement Single-domain antibody binding studies based on SPR (surface plasmon resonance) technology were conducted on a Biacore T200 optical biosensor instrument (GE Healthcare). Recombinant NB-6xHis was captured on a nitrogen-triacetic acid (NTA) sensor chip in HBS-P+ buffer (10 mM HEPES pH 7.5, 150 mM NaCl, and 0.05% surfactant P20). For the immobilization strategy, the flow cell was loaded with nickel solution (Sigma Aldrich) to saturate the NTA surface with Ni2+, and additional washing was performed with run buffer containing 3 mM EDTA after nickel implantation. The His-tagged single-domain antibody was diluted in HBS-P+ buffer and added to the flow cell at a concentration of 50 μg / mL.

[0176] Because the single-domain antibody is smaller than its target trypsin-3 protein, Nb-6His was captured on the sensor chip at 4°C. Active trypsin-3 or a trypsin-3 precursor was used as the analyte. The analyte was injected sequentially in single cycles at concentration gradients from 9 nM to 500 nM, without regenerating the sensor chip between injections. The analyte was diluted in HBSC-P+ buffer (HBS-P containing 10 mM CaCl2). Four flow cells (FCs) of the sensor chip were used: one (FC1) was used to monitor nonspecific binding and provide background correction for the analysis, while the other three flow cells (FC2, FC3, and FC4) were immobilized with Nb-6His for measurement. Insensitive Nb was used as a negative control (see Bery, Cell Chem Biol 2019). The true binding response was obtained by subtracting the FC1 flow cell from the flow cell immobilized with trypsin-3. The kinetic constants (kon, koff, KD = kon / koff) were calculated using BIAevaluation 4.0.1 software, and the 1 / 1 Langmuir binding model was selected. This model determines the binding constant (kon) and accounts for dissociation that occurs during the binding phase. Therefore, the calculated values ​​are not necessarily directly related to the apparent slope of the sensor map.

[0177] PC3 processing PC3 cells (ATCCCRL-1435 provided by Dr. Olivier Cuvillier, IPBS, Toulouse, France) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 1× penicillin / streptomycin, and 1× non-essential amino acid solution (ThermoFisher #11140050) at 37°C in a humidified incubator containing 5% CO2. For morphological assays, 10,000 cells were seeded into wells containing glass coverslips. After 3 days, the DMEM medium was removed, and the cells were treated for 24 hours. After treatment, the cells were washed once with PBS 1X. Very gentle washing was used to prevent cell detachment. Cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton for 10 minutes. Cover slides were incubated with 1 / 200 Alexa Fluor 647 phalloidin (#A22287, ThermoFisher) solution at room temperature for 30 minutes to stain cell membranes. The coverslips were briefly rinsed twice and mounted with Prolong Gold anti-fluorescence quenching mounting medium containing DAPI (P36935, Invitrogen). The roundness index of each cell was determined using ImageJ software. At least 200 cells were analyzed under each condition, and the experiment was repeated more than three times.

[0178] Computer Analysis To determine the linker length required for simultaneous binding of two nanobodies to trypsin-3, computer simulations were performed using Pymol 2.5.1 software (Schrodinger, LLC) and a three-dimensional structural model of trypsin-3 obtained from 2.38 Å X-ray diffraction studies (PDB 3L3T DOI:10.2210 / pdb3L3T / pdb) (Salameh, MA, Soares, AS, Navaneetham, D., Sinha, D., Walsh, PN, Radisky, ES. Determinants of affinity and proteolytic stability in interactions of Kunitzfamily protease inhibitors with mesotrypsin. (2010) J Biol Chem 285: 36884-36896). The nanobody structures were modeled using the Swiss model (https: / / swissmodel.expasy.org / ) with 50 templates, and the resulting structures were compared with those obtained using α-sheets. This verified the reliability of the structure. All superpositions and structural diagrams were created using the graphical software PyMOL. The minimum length was calculated based on the maximum distance between two opposing epitopes of trypsin-3. The enzyme exhibits an oval structure, and the appropriate linker length was estimated by determining multiple distances.

[0179] Statistical analysis Analysis was performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). Multiple comparisons were performed using repeated measures one-way ANOVA, followed by the Kruskal-Wallis test. Statistical significance was considered to be ≤0.05.

[0180] result Isolation of trypsin-3 specific single-domain antibody To achieve high selectivity for trypsin-3, this disclosure uses a highly diverse synthetic single-domain antibody library (see Moutel et al., 2016, eLife) for phage display screening. To avoid diversity bias caused by the protease activity of natural antigens in the early stages of screening and to enrich a large number of single-domain antibodies that react with trypsin-3, this disclosure prepares recombinant mutant trypsin-1, trypsin-2, and trypsin-3 as inactive mature proteases and establishes a reduction screening protocol. Therefore, the phage display library is screened for removal from inactive trypsin-1 and trypsin-2. Then, in two rounds of screening, inactive trypsin-3 is positively screened in the presence of excess inactive trypsin-1 and trypsin-2. The final round of screening uses wild-type active trypsin-3 to preferentially enrich single-domain antibodies with enzyme activity inhibition. The selected single-domain antibodies are screened by ELISA to evaluate their detection capabilities for trypsin-1, trypsin-2, trypsinogen-3, and active trypsin-3. Seventeen clones exhibiting significant specificity for active trypsin-3 were sequenced, identifying five distinct single-domain antibodies (NT3-1, NT3-3, NT3-7, NT3-12, and NT3-16). To confirm their specificity and affinity for active trypsin-3, these five single-domain antibodies were purified and further characterized. First, binding experiments were performed on these five candidate antibodies based on surface plasmon resonance (SPR) analysis (Table 6). The single-domain antibodies were immobilized on a sensor chip, and their binding ability was detected in real-time by injecting different concentrations of recombinant trypsin-3. Three parameters were analyzed: binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD). All five single-domain antibodies were able to bind to wild-type or catalytically inactivated mutants of mature human Trypsin-3, but binding to the precursor form of Trypsin-3 (data not shown) or mature Trypsin-1 and Trypsin-2 isoforms was not observed. This disclosure observes a typical binding mode of sdAbs with very rapid binding and dissociation rates in a dose-dependent manner. Five NT3s exhibit very interesting affinity for trypsin-3, with KD values ​​less than 10 nM. Although NT3-16 has a high binding rate constant, its dissociation rate with the protease is even faster (Table 6). The stoichiometric ratio of the interaction between active trypsin-3 and each single-domain antibody is 1:1.

[0181] Characterization of trypsin-3 single-domain antibody as a protease inhibitor To evaluate the inhibitory activity of each single-domain antibody against trypsin-3, this disclosure performed an enzyme inhibition assay, monitoring the enzymatic cleavage of the fluorescent peptide substrate by trypsin-3 in the presence of different concentrations of single-domain antibodies. Conversely, in the binding assay, only NT3-7 and NT3-12 showed inhibitory activity against active trypsin-3. To quantify the ability of NT3-7 and NT3-12 to regulate trypsin-3 activity, this disclosure monitored the inhibition of trypsin-3 activity by increasing the concentration of the single-domain antibody. The half-maximal inhibitory concentration (IC50) obtained by semi-logarithmic plotting was in the low micromolar range (Figure 1). Compared with NT3-12, NT3-7 had a significantly lower IC50 value (IC50 values ​​of 1.5 ± 0.3 μM and 8.81 ± 1.45 μM, respectively) (Figure 1a-b).

[0182] To characterize the inhibitory mode of the single-domain antibody, competitive inhibition experiments were conducted using different concentrations of inhibitor and substrate to detect protease activity in the reaction mixture. Data obtained from the reaction progress curves were used to determine the interaction mode between the inhibitor and trypsin-3 (Figure 1c-d). The presence of NT3-12 decreased the KM value, while Vmax was similar to that in the presence of protease alone. NT3-12 interacted with active trypsin-3 as a competitive inhibitor, with an apparent inhibition constant KI of 535 nM. In contrast, the NT3-7 single-domain antibody exhibited a different inhibitory mode, appearing to antagonize protease activity in a non-competitive inhibitory manner (simultaneously altering KM and Vmax), with an apparent KI of 46 nM.

[0183] The antigen-binding site of a single-domain antibody consists of three complementarity-determining regions (CDRs) supported by framework residues. In the context of this study, the framework of all single-domain antibodies is identical; the diversity of antigen-binding sites is provided by the CDR regions. When the modeling structures of each strong inhibitor antibody (NT3-7 or NT3-12) were superimposed with those of antibodies with affinity for trypsin-3 (NT3-1, NT3-3, NT3-16) or lack thereof (NT3-2, NT3-5), it was observed that only antibodies with affinity for trypsin-3 exhibited identical folding in CDR1 and CDR2. The ability to inhibit trypsin-3 appears to depend on the shape of the CDR3 ring (data not shown).

[0184] Then, the ability of single-domain antibodies to selectively inhibit the activity of other proteases was evaluated against a group of proteases with similar trypsin-like activity (trypsin-1, trypsin-2, thrombin, KLK1, KLK3, and KLK5). Inhibition assays showed that at molecular concentrations of sdAbs capable of inhibiting 42% of trypsin-3 activity (inhibitor / protease = 1000), both NT3s were poor inhibitors of other trypsin-like proteases (maximum inhibition of KLK5 = 12%). Adding Nb to the trypsin-1 reaction mixture induced a decrease in protease activity, but this decrease was not specific, as the addition of unrelated Nb also induced a decrease in protease activity, and this decrease was independent of the Nb dose (data not shown). In the same manner, NT3-7 and NT3-12 had no effect on KLK3 and thrombin. Figure 2 Therefore, both of these single-domain antibodies are highly selective trypsin-3 inhibitors.

[0185] Development of a dual-epitope trypsin-3 single-domain antibody.

[0186] To enhance the affinity of trypsin-3 single-domain antibodies, a biepisode dimer was constructed by fusing a trypsin-3 inhibitor (NT3-7 or NT3-12) with another single-domain antibody exhibiting high affinity for trypsin-3 (Figure 3A). The potential for simultaneous binding of both single-domain antibodies to trypsin-3 was first assessed using an ELISA-sandwich assay. The HIS-tagged trypsin-3 inhibitor single-domain antibody was immobilized on the surface of microplate wells as a capture fraction, then incubated first with trypsin-3, and further incubated with a candidate FLAG-tagged single-domain antibody as the trypsin-3 detection molecule. After washing, the amount of bound single-domain antibody was detected using HRP-conjugated anti-FLAG antibody. Under these conditions, three single-domain antibodies were able to detect trypsin-3. NT3-1, NT3-12, and NT3-16 can bind to trypsin-3 simultaneously with NT3-7, and NT3-1 can bind to the protease simultaneously with the competitive inhibitor NT3-12 (Figure 3B), indicating that the dual epitope combination is feasible.

[0187] To optimize the linker that facilitates the interaction between two single-domain antibodies on trypsin-3, computer simulations were performed using a 3D model of trypsin-3. Trypsin-3 is an oval protein, and the distance between the two furthest points was calculated (data not shown). Furthermore, the linker sequence was designed to ensure the flexibility of the biepisode bioactive molecule while avoiding the use of known proteases to predict the amino acids at the sensitive sites. Then, an empirically designed linker with a potential length of 70.5 Å was developed, primarily based on a glycine extension consisting mainly of glycine repeats and serine or glutamic acid residues as solubility regulators (Chen, Zaro, & Shen, 2013) (data not shown).

[0188] Five combinations were generated in the heterologous system and their inhibitory activity was tested. Surprisingly, the NT3-7 dimer and NT3-7+NT3-1 did not inhibit trypsin-3 (data not shown). The fusion of NT3-7 with the high-affinity NT3-16 showed only a weak inhibitory effect. Figure 4A In contrast, the combined use of two inhibitors (NT3-7 and NT3-12) significantly improved the inhibitory effect on Nb. Figure 4A Compared to NT3-7 (IC50 = 1.5 µM), this dual-epitope single-domain antibody exhibits a very low IC50 value (0.043 µM) and primarily functions as a competitive inhibitor of trypsin-3. Another fusion orientation, NT3-12 linked to NT3-7, demonstrates the same inhibitory behavior against trypsin-3. Figure 4A ).

[0189] To comprehensively characterize the properties of the dual epitope trypsin-3 inhibitor, its binding kinetics were evaluated using SPR. The KD value of the dual epitope NT3-7 / 12 was more than 30-fold lower than that of NT3-7 alone. Figure 4B (Table 6) The values ​​were 0.046 nM and 1.31 nM, respectively, indicating that NT3-7 / 12 has a very high affinity for active trypsin-3. The interaction between the target protease and the inhibitor was significantly enhanced. As expected, the dissociation of the complex on the sensor chip was very slow ( Figure 4B Furthermore, the fusion of two trypsin-3 inhibitory single-domain antibodies improved selectivity for the target protease. This dual-epitope nanobody inhibited active trypsin-3 only in a competitive mode, with no further inhibition of KLK5 observed (data not shown).

[0190] Intracellular efficacy of biepisotope trypsin-3 single-domain antibodies To evaluate the ability of NT3-7 / 12 to control trypsin-3 activity in complex environments, PC3 cells were used. These cells exhibit a natural "fibroblast" morphology. Previous studies have shown that gene knockout of trypsin-3 expression in these cells induces a change in phenotype to roundness (Cohen et al., 2016). Using this characteristic, cell roundness was evaluated to assess the inhibitory capacity of single-domain antibodies. PC3 cells were treated with different concentrations of NT3-7 / 12 or NT3-7 / 7 (as a negative control, a non-inhibitory bimodal epitope Nb). Adding NT3-7 / 7 to the PC3 cell culture medium had no effect on the cells, while increasing the concentration of NT3-7 / 12 induced a change in cell shape, with elongated cells becoming rounder. Figure 5A The half-maximal effective concentration (EC50) was calculated to be 642 nM based on the dose-response relationship of cell roundness parameters under NT3-7 / 12 treatment. Therefore, this dual-epitope nanobody can tightly regulate the activity of secretory trypsin-3 in challenging environments.

[0191] Table 6: Kinetic parameters of the interaction between trypsin-3 and single-domain antibodies

[0192] Example 2 To evaluate the ability of NT3-7 or NT3-7 / 12 to control trypsin-3 activity in a human setting, colonic biopsy sections from patients with irritable bowel syndrome (IBS) were pre-incubated with 10 µM of anti-trypsin-3 single-domain antibody and in situ zymography was performed according to the method described by Rolland-Fourcade et al. (Rolland-Fourcade et al. Epithelial expression and function of Trypsin-3 in irritable bowel syndrome. Gut 2017). Under control conditions, the proteolytic activity detected at the epithelial cell level was significantly reduced in the presence of anti-trypsin-3 single-domain antibody. Therefore, anti-trypsin-3 single-domain antibody can inhibit the overactive release of trypsin-3 from human epithelial cells under IBS conditions.

[0193] Example 3 Given that trypsin-3 is considered to play a key role in migration (Hockla, 2012), the ability of NT3-7 / 12 to control cell migration was evaluated using a PC3 cell wound healing assay. NT3-7 / 12 was added to the culture medium concurrently with scratch creation. The NT3-7 / 12 inhibitor reduced the migration rate of PC3 cells in a dose-dependent manner. Figure 5BThe addition of NT3-7 / 7 had no effect on the migration ability of PC3 cells, indicating that this dual-epitope nanobody can inhibit the migration of metastatic prostate cancer cells by regulating trypsin-3 activity.

[0194] Table 7: Available amino acid sequences for carrying out the present invention

[0195]

[0196]

[0197]

[0198] References: Throughout this application, various references describe the current state of the art to which this invention pertains. All disclosures in the aforementioned references are incorporated herein by reference.

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Claims

1. An antitrypsin-3 single-domain antibody (sdAb), wherein, The antibody specifically binds to human trypsin-3 protein and does not bind to trypsin-3 precursors and / or mature trypsin-2 and / or trypsin-1 isoforms.

2. The antitrypsin-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 exhibits inhibitory ability against active human trypsin-3 (neutralizing antibody); (ii) Its KD for binding to human active trypsin-3 protein is 200 nM or less, 100 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, or 0.05 nM or less.

3. The antitrypsin-3 single-domain antibody according to claim 1 or 2, wherein, The single-domain antibody contains (a) CDR1 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 2, CDR2 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 3, and CDR3 having the NT3-7 sdAb sequence as shown in SEQ ID NO: 4; or (b) CDR1 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 6, CDR2 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 7, and CDR3 having the NT3-12 sdAb sequence as shown in SEQ ID NO: 8; or (c) CDR1 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 10, CDR2 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 11, and CDR3 having the NT3-1 sdAb sequence as shown in SEQ ID NO: 12; or (d) CDR1 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 14, CDR2 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 15, and CDR3 having the NT3-3 sdAb sequence as shown in SEQ ID NO: 16; or (e) CDR1 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 18, CDR2 having the NT3-16 sdAb sequence as shown in SEQ ID NO: 19, and CDR3 having the NT3-16 sdAb sequence as shown in SEQ ID NO:

20.

4. The antitrypsin-3 single-domain antibody according to any one of claims 1 to 3, wherein, The single-domain antibody comprises: (a) A heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 1 (NT3-7); or (b) A heavy chain variable region (VH) having at least 70% identity with the sequence shown in SEQ ID NO: 5 (NT3-12); or (c) A heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 9 (NT3-1); or (d) A heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 13 (NT3-3); or (e) Heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 17 (NT3-16).

5. The antitrypsin-3 single-domain antibody according to any one of claims 1 to 4, wherein, The single-domain antibody has a heavy chain variable region (VH) sequence as shown in SEQ ID NO: 1 ("NT3-7"), SEQ ID NO: 5 ("NT3-12"), SEQ ID NO: 9 ("NT3-1"), or SEQ ID NO: 13 ("NT3-3"), or SEQ ID NO: 17 ("NT3-16").

6. The antitrypsin-3 single-domain antibody according to claim 5, wherein, The single-domain antibody has a heavy chain variable region (VH) sequence as shown in SEQ ID NO: 1 ("NT3-7") or SEQ ID NO: 5 ("NT3-12").

7. A cross-competitive single-domain antibody that cross-competitively binds to trypsin-3 with any one of the single-domain antibodies 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, comprising at least two single-domain antibodies according to any one of claims 1 to 5.

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

11. The polypeptide according to any one of claims 8 to 10, comprising the sequence of the heavy chain variable region (VH) as shown in SEQ ID NO: 1 ("NT3-7") and the sequence of the heavy chain variable region (VH) as shown in SEQ ID NO: 5 ("NT3-12").

12. A nucleic acid sequence encoding an antitrypsin-3 single-domain antibody as described in any one of claims 1 to 6, or a cross-competitive single-domain antibody as described in claim 7, or a polypeptide as described in any one of claims 8 to 11.

13. A vector comprising the nucleic acid sequence according to claim 12.

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

15. The pharmaceutical composition according to claim 14, used as a medicine.

16. The pharmaceutical composition of claim 15, for treating intestinal diseases related to intestinal permeability, said intestinal diseases selected from the list of: irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), celiac disease, or pouchitis.

17. The pharmaceutical composition according to claim 15, for the treatment of cancer, particularly cancers associated with trypsin-3.

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