Immunoglobulin single domain antibodies for mucosal vaccine delivery

Abstract

The present invention relates to single domain antibodies that specifically bind to aminopeptidase N (APN), and more particularly to polypeptides and nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to compositions comprising such polypeptides for prophylactic, therapeutic or diagnostic purposes, and especially to pharmaceutical compositions. In particular, the single domain antibodies of the invention are capable of targeting moieties to mucosal surfaces.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to single domain antibodies that specifically bind to aminopeptidase N (APN) and more particularly to polypeptides and nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to compositions comprising such polypeptides for prophylactic, therapeutic or diagnostic purposes, and especially to pharmaceutical compositions. In particular, the single domain antibodies of the invention allow for targeting moieties to mucosal surfaces. [Background technology]

[0002] Background of the Invention The majority of pathogens and foreign harmful substances enter the body through the mucosal surfaces of the respiratory, gastrointestinal (GI), and genitourinary tracts, and therefore mucosal immunity is paramount for combating infection. Needle-free oral vaccine delivery is currently the most attractive route of administration for protection against enteric pathogens because it offers many advantages, such as ease of administration, no risk of bloodborne infection, and practicality for large-scale vaccination. In contrast to subunit vaccines, only live-attenuated vaccines or inactivated pathogen particles have been found to be effective in stimulating efficient immune responses. However, due to safety issues associated with orally delivered live-attenuated or inactivated pathogens, only a handful of such vaccines have been approved. On the other hand, subunit vaccines are considered safe because they do not contain pathogen life components. However, poor transport of vaccine antigens across the intestinal epithelium to reach the underlying immune-inducing site, leading to poor immune responses, poses a major hurdle to the development of oral vaccines. Enterocytes, or absorptive villous epithelial cells, comprise 90% of all intestinal epithelium and possess the capabilities of phagocytosis and transcytosis to transport intestinal pathogens or macromolecules across the epithelial barrier [1]. Therefore, antigen targeting transcytosis receptors in enterocytes is an attractive approach for vaccine delivery and to induce strong mucosal and systemic immune responses against intestinal pathogens [2].

[0003] Recently, aminopeptidase N (APN) has been identified as a receptor for F4 fimbriae expressed on various cells, including small intestinal enterocytes and antigen-presenting cells (APCs), but not on epithelial cells in other parts of the GI tract [3]. Oral immunization with an APN-specific polyclonal antibody triggered mucosal immunity [3, WO09 / 103555]. Furthermore, functionalization of microparticles with an APN-specific mouse monoclonal antibody also increased the uptake of these particles by the intestinal epithelium and triggered a systemic immune response [4]. These findings suggest that APN-specific delivery agents conjugated with vaccine antigens may be an attractive strategy for eliciting stronger intestinal immune responses.

[0004] To date, several targeting mechanisms have been identified, including antibodies, cell-mediated targeting (DC immunotherapy, T cell adoptive transfer), and chemical conjugation of antigens to small molecules such as glycans and amino acids [5]. Due to their strong affinity and specificity, antibodies are ideal proteins to which antigens of interest can be genetically fused to target APN-expressing intestinal cells. Various antibody formats, such as monoclonal antibodies (mAbs), single-chain variable fragments (scFvs), or heavy-chain-only antibody variable domains (VHHs, also known as Nanobodies®), can be used. While traditional mAbs were originally preferred as targeting vehicles, their production when fused to antigens was cumbersome and time-consuming. Additionally, their structural complexity precludes their use as potential ligands. Therefore, other simpler formats, such as scFvs and VHHs, have been considered alternative approaches to full-sized antibodies.

[0005] Although advances in antibody engineering have made it possible to easily design and clone scFv genes based on the targeting capabilities of mAbs, the stability and accumulation of synthetic scFv proteins vary greatly depending on several factors, including the unpredictable length of the linker required for correct folding and assembly of the two variable domains, which often limits the application of scFv targeting [6, 7]. On the other hand, VHH immunoglobulin domains are variable fragments of only 15 kDa derived from camelid heavy-chain-only antibodies [8]. Unlike scFvs, VHHs contain their antigen-binding properties in a single domain rather than in the two variable domains of conventional antibodies and resulting scFvs, while retaining similar affinity. Due to their unique properties, such as small size, ease of production, good thermal stability, and efficient tissue penetration, VHHs have attracted great interest for various applications compared to classical antibodies [9-11]. The present invention provides, by way of example, VHHs and their application as carriers for targeted delivery to the gastrointestinal epithelium to induce strong mucosal immune responses. Summary of the Invention

[0006] SUMMARY OF THE INVENTION The present invention has identified a family of VHHs that can be stably produced at high levels. This family of VHHs not only binds to APN, but is also efficiently endocytosed by cell lines and in the loops of the porcine gastrointestinal tract under physiological conditions, and, notably, is capable of eliciting systemic and intestinal IgA responses, particularly after oral administration. The polypeptides of the present invention can be used as delivery vehicles to target APN receptors and efficiently deliver molecules to mucosae that specifically express APN, more specifically to the (small) intestine. Furthermore, the polypeptides of the present invention cross epithelial barriers after oral administration to pigs, demonstrating their usefulness as carriers for targeting heterologous compounds / antigens to the intestinal mucosal immune system.

[0007] In one embodiment, a polypeptide of the invention comprises at least one immunoglobulin single variable domain (ISVD), wherein said ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is SEQ ID NO: 1, and an amino acid sequence having 1, 2 or 3 amino acid difference(s), particularly 2 amino acid differences, more particularly 1 amino acid difference, from SEQ ID NO: 1 Selected from the group consisting of; (ii) CDR2 is SEQ ID NO: 3, and an amino acid sequence having 1, 2 or 3 amino acid difference(s), particularly 2 amino acid differences, more particularly 1 amino acid difference, from SEQ ID NO: 3 Selected from the group consisting of; and (iii) CDR3 is SEQ ID NO: 4, and Amino acid sequences having 1, 2 or 3 amino acid difference(s), particularly 2 amino acid differences, more particularly 1 amino acid difference, from SEQ ID NO: 4 is selected from the group consisting of:

[0008] More specifically, (i) CDR1 is selected from SEQ ID NO: 1 or 2; (ii) CDR2 is SEQ ID NO: 3; and (iii) CDR3 is selected from SEQ ID NO: 4 or 5. Particular polypeptides according to the invention are as follows: - CDR1 is SEQ ID NO: 1, CDR2 is SEQ ID NO: 3, and CDR3 is SEQ ID NO: 4; or - CDR1 is SEQ ID NO: 2, CDR2 is SEQ ID NO: 3, and CDR3 is SEQ ID NO: 5, The CDR1, CDR2 and CDR3 combinations are selected from: More particularly, an ISVD according to the present invention consists of or consists essentially of four framework regions (FR1 to FR4, respectively) and complementarity determining regions CDR1, CDR2 and CDR3 as provided herein.

[0009] In one aspect, the present invention provides a polypeptide comprising or consisting of SEQ ID NO:6, or a polypeptide having at least 80% sequence identity to SEQ ID NO:6, such as, for example, SEQ ID NO:7 or SEQ ID NO:8. In a further embodiment, an APN-binding construct is provided comprising at least one polypeptide of the present invention. The construct may contain another moiety linked to the immunoglobulin single variable domain. The additional moiety may or may not bind to APN. In a specific embodiment, the construct further comprises an Fc domain and / or a further diagnostic or therapeutic moiety, such as a biologically active compound.

[0010] In another embodiment, the immunoglobulin single variable domains are not provided per se, but as nucleic acids, i.e., isolated or recombinant nucleic acid molecules encoding the polypeptides as described herein. Furthermore, vectors or host cells containing such nucleic acids are provided. Typically, such host cells will have been transformed or transfected with the nucleic acid. A particular use envisioned for these host cells is the production of the immunoglobulin single variable domains or polypeptides of the invention. Thus, host cells transformed or transfected with nucleic acid molecules encoding the polypeptides provided herein can be used for the production of immunoglobulin single variable domains.

[0011] According to a further aspect, the polypeptides, nucleic acids or constructs provided herein are for use in medicine, especially for use as medicines in either human or veterinary medicine. According to another embodiment, the polypeptides (or nucleic acids encoding them) are provided for use in the therapeutic treatment or prevention of (gastro)intestinal diseases.

[0012] The polypeptides of the invention may be provided as proteins (as immunoglobulin single variable domains, as part of an APN-binding construct, chimeric molecule or pharmaceutical composition) or may be administered as nucleic acid molecules encoding said immunoglobulin single variable domains or as vectors comprising such nucleic acid molecules. Various routes of administration may be envisaged. By way of non-limiting example, the polypeptides may be administered systemically, orally or intranasally, but particularly orally. When the immunoglobulin single variable domain is provided as a nucleic acid or vector, it is specifically envisaged that the immunoglobulin single variable domain is administered via gene therapy. Particular embodiments provide polypeptides, constructs, chimeric molecules or pharmaceutical compositions for use in vaccination, particularly mucosal vaccination.

[0013] In one aspect, the present invention provides a pharmaceutical composition comprising a polypeptide, construct or chimeric molecule, nucleic acid or host cell as described herein and a pharmaceutically acceptable carrier, excipient and / or diluent. In particular, the pharmaceutical composition is a vaccine, more in particular an oral vaccine, i.e. a vaccine suitably formulated for oral delivery. The present invention further encompasses a method of treating or preventing an intestinal disease or disorder in a subject, the method comprising administering to the subject a polypeptide, construct, chimeric molecule or pharmaceutical composition in an amount effective to treat, reduce or prevent at least one symptom of the disease or disorder.

[0014] In another aspect, the present invention relates to the polypeptide, construct or chimeric molecule, nucleic acid or host cell as described herein for diagnostic use, for example for use in bioimaging or for use in competitive assay.In addition, polypeptide, construct or chimeric molecule can be used in the in vitro method for identifying and obtaining the compound or organism (for example, virus) that can cross the mucosal barrier in a subject, said method comprising: a) incubating the source, cell or cell line that contains APN or its functional fragment with the compound or organism to be tested; and b) determining the ability of said molecule to compete with polypeptide or chimeric molecule as provided herein. Also provided is a method for producing a polypeptide of the invention, the method comprising the steps of: - introducing a nucleic acid encoding the polypeptide into an expression system, in particular Pichia pastoris, and -purifying the expressed polypeptide Includes. [Brief explanation of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1: Schematic diagram of the BioXp™ 3200 system-based Gibson cloning strategy for efficiently obtaining VHH-MG (heavy-chain-only antibody variable domain fused to the Fc domain of mouse IgG) fusions. The 40 bp at the ends of the VHH-MG fusion fragment are homologous to the ends of the SapI-linearized pKaiGG vector. MG represents the Fc domain of mouse IgG. The VHH-MG fragment was synthesized and ligated into SapI-digested pKaiGG in an overnight run in the BioXP system. DH5α cells were transformed with the ligation sample, and transformants were screened in Zeocin (Zeo)-supplemented medium. Subsequently, DNA was isolated from four randomly selected clones and analyzed by restriction digestion or colony PCR and sequencing to screen for positive clones. (b) Efficiency of obtaining error-free clones via the BioXP cloning system. The probability of obtaining an error-free clone was 77% when screening one clone per construct via sequencing, and 97% when screening two clones.

[0016] [Figure 2]Figure 2: Selection of APN-specific VHH-MGs via ELISA and flow cytometry. Clones are arranged according to their family and separated by dotted lines. (a) ELISA plates were coated with APN and incubated with culture medium. Binding of VHH-MGs to APN is depicted as the OD value of the colorimetric reaction. (b) Flow cytometry screening for VHH-MGs that bind to full-length APN expressed in the membranes of APN-transfected BHK21 (BHK21-APN, gray bars) and APN-transfected IPEC-J2 (IPEC-J2-APN, black bars). IMM013 (mouse IgG1) is a positive control. IgG1 isotypes, V2-MG and D3-MG, are negative controls. V2-MG and D3-MG fusions contain VHHs (V2 and D3, respectively) that bind to unrelated targets. The graph shows the mean fluorescence intensity (MFI).

[0017] [Figure 3] Figure 3: Purification of various VHH-MG fusions. (a) Size-exclusion chromatography (SEC) profiles of six various VHH-MG fusions after protein A purification, each labeled with its name in the upper right corner. Arrows indicate the positions corresponding to the monomeric VHH-MG fusions assembled by disulfide bridges between two VHH-MG polypeptides (each ∼40 kDa) and the molecular weight of ∼80 kDa. (b) SDS-PAGE analysis of pooled fractions of the 80 kDa peak after SEC on a 4-20% polyacrylamide gel under both reducing (left) and non-reducing (right) conditions. The expected positions of the intact, full-length polypeptides are indicated by arrowheads for monovalent VHH-MG (∼40 kDa) under reducing conditions and by arrows for bivalent VHH-MG (∼80 kDa) under non-reducing conditions. M, molecular weight marker (kDa). (Yields - 3L94-MG: 2.7 mg / L; 2L48-MG: 2.3 mg / L; 2L69-MG: 1.9 mg / L; 2L65-MG: 5.9 mg / L; 2L22-MG: 8 mg / L; 2L46-MG: 1.4 mg / L).

[0018] [Figure 4] Figure 4: Screening of VHHs binding to APN-expressing cell lines. Crude periplasmic extracts are prepared from TG1 cells harboring recombinant phagemids from the library. 28 APN-binding VHH candidates were screened by flow cytometry in APN-expressing cell lines. Clones were grouped according to their similarity in CDR3 sequences; the numbers at the top between the dotted lines refer to the families to which the various clones belong. The Y-axis represents the ratio of median fluorescence intensity (MFI) of APN-transfected (APN) and parental non-transfected BHK21 cells (Parent).

[0019] [Figure 5] Figure 5: Binding analysis of purified VHH-MG fusions and APN-mediated endocytosis. (a) APN-binding ELISA of purified VHH-MG fusions. A three-fold dilution series of purified VHH-MG was incubated in APN-coated microtiter plates and probed with anti-mouse IgG conjugated with horseradish peroxidase. Binding of VHH-MG to immobilized antigen is depicted as OD (492 nm) values. IMM013 was a positive control, and GBP-MG was a negative control. The background threshold was determined as twice the OD (492 nm) value of the negative control. (b) Binding of VHH-MG fusions to APN-expressing cells was determined by flow cytometry. The graph shows the number of APN-positive cells binding to VHH-MG at the indicated concentrations (μg / ml).

[0020] [Figure 6]Figure 6: Oral immunization of piglets with VHH-MG triggers circulating and small intestinal antibody responses. (a) Design of the oral immunization experiment. (b) Mouse IgG2a-specific serum IgG and IgA responses upon oral immunization of piglets (n=4 / group) with 1 mg of VHH-MG 2L65 and 3L94 and an equimolar amount of irrelevant mouse IgG2a. dppi: days after primary immunization; OD: optical density. Data are expressed as mean+sd. *, p<0.05 vs. IgG2a (Friedman). (c) Amounts of circulating mouse IgG2a-specific IgG- and IgA-secreting cells at the indicated time points. ASC: antibody-secreting cells. **, p<0.01 vs. day 0; Δ, p<0.05 vs. IgG2a (Friedman). (d) Amounts of mouse IgG2a-specific IgG- and IgA-secreting cells in the small intestine at d28 after primary immunization. MLN: mesenteric lymph nodes; JLP: jejunal lamina propria; JPP: jejunal Peyer's patches; ILP: ileal lamina propria; IPP: ileal Peyer's patches. *, p<0.05 (Holm-Sidak).

[0021] [Figure 7] Figure 7: Expression of various variants of APN-specific VHH-MG fusions in Pichia pastoris. Culture media of yeast transformants (four colonies for each construct) were screened by Coomassie staining and Western blot analysis to identify the highest-expressing clones. Here, only one highly expressing clone for each construct is shown, and they are grouped according to their CDR3 similarity; the numbers between the dotted lines represent their family membership. Protein expression was induced with 1% methanol for 48 h. Cultures were harvested, and supernatants were analyzed by SDS-PAGE (top) and Western blot (bottom). "V2-MG Pure" was used as a loading control, and 500 ng of the sample was loaded for Coomassie staining and 200 ng for Western blot.

[0022] [Figure 8]Figure 8: Representative amino acid and nucleic acid sequences of polypeptides of the invention. CDRs are underlined. [Figure 9] Figure 9: Amino acid and nucleic acid sequences of the VHH-Fc mouse IgG2a fusion construct (2L65-MG fusion). The sequence of 2L65VHH is in bold. The hinge sequence is underlined. [Figure 10] Figure 10: (a) Amino acid sequence of a portion of porcine IgA (AAA65943.1; U12594.1); (b) Hinge-CH2-CH3 fragment of porcine IgA; (c) Amino acid sequence of a portion of porcine IgG3 (EU372658.1); (d) CH1-hinge-CH2-CH3 fragment of IgG3 (CH1 domain starts at position 5). [Figure 11] Figure 11: APN-specific VHHs are internalized by BHK-APN cells.

[0023] Description of the invention Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art and will be clear to one of ordinary skill in the art. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that could be modified or substituted for the method described herein.

[0024] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. The term "and / or," whenever used herein, encompasses the meanings of "and," "or," and "all or any other combination of the elements connected by said term." The terms "about" or "approximately," as used herein, mean within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range. Throughout this specification and the claims that follow, unless the context otherwise requires, the words "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be substituted by the terms "containing" or "including," or sometimes, as used herein, by the term "having."

[0025] The term "sequence", as used herein (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence"), should generally be understood to encompass both related amino acid sequences as well as nucleic acid or nucleotide sequences encoding the same, unless a more restricted interpretation is required. A nucleic acid or amino acid sequence is considered to be "(in) (essentially) isolated" when it is separated from at least one other component with which it is most often associated in the source or medium - e.g., compared to the reaction or culture medium from which it was obtained - such as another nucleic acid or amino acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or minor component.

[0026] For purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing the number of nucleotides in the first nucleotide sequence that are identical to the nucleotide at the corresponding position in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence—compared to the first nucleotide sequence—is considered a difference of a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings.

[0027] For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" (herein also referred to as "amino acid identity") between a first amino acid sequence and a second amino acid sequence may be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residue at the corresponding position in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence, and multiplying by 100%, with each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence—being considered a difference of a single amino acid residue (position), i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using known computer algorithms, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.

[0028] In determining the degree of sequence identity between two amino acid sequences, those skilled in the art may also consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and essentially has little or no effect on the function, activity or other biological properties of a polypeptide. Such conservative substitutions are preferably those in which one amino acid in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0029] Specifically preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu. Any amino acid substitutions applied to the polypeptides described herein may also be based on an analysis of the frequency of amino acid variations between homologous proteins of different species.

[0030] Unless otherwise specified, the terms "immunoglobulin" and "immunoglobulin sequence" are used generically to encompass both full-size antibodies. An "immunoglobulin single variable domain" or "ISVD" is an antibody fragment consisting of a single variable antibody domain. Like a whole antibody, it can selectively bind to a specific antigen. With a molecular weight of only 12-18 kDa, an ISVD is much smaller than a conventional antibody (150-160 kDa) composed of two heavy and two light protein chains, and even smaller than a Fab fragment (approximately 50 kDa, one light chain and half a heavy chain) and a single-chain variable fragment (approximately 25 kDa, two variable domains, one from a light chain and one from a heavy chain). Generally, an ISVD will have an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), preferably according to the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antigen-binding site of an ISVD is formed by only three CDRs. As used herein, the term "ISVD" encompasses variable domains of camelid heavy chain antibodies (VHHs), also referred to as Nanobodies®, domain antibodies (dAbs), and ISVDs derived from sharks (IgNAR domains). Preferred framework sequences are outlined, for example, in Figure 8 and can be used in the ISVDs of the present invention. Preferably, the CDRs depicted in Table 2 are compatible with each framework region of the same ISVD construct.

[0031] The term "immunoglobulin single variable domain" is used interchangeably with "single variable domain". Thus, a single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, so long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). In one embodiment, the immunoglobulin single variable domain is a VHH sequence.

[0032] The APN binders of the present invention may be immunoglobulins, such as immunoglobulin single variable domains, in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e. from a suitable species of Camelidae), or synthetic or semi-synthetic amino acid sequences, including but not limited to, "humanized" VHH sequences, "camelized" immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), and Nanobodies®, obtained by techniques such as affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CD grafting, veneering, combination of fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for modifying immunoglobulin sequences that are well known to those skilled in the art; or any suitable combination of any of the above.

[0033] More particularly, the present invention provides polypeptides which specifically bind to APN and which comprise at least one immunoglobulin single variable domain which is an amino acid sequence with the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively. In one embodiment of the invention, the ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 6 to 8, and in particular with SEQ ID NO: 6 (see Figure 8 - CDRs are underlined).

[0034] As used herein, "represented by," in the context of any SEQ ID NO:, is equivalent to "comprising" or "consisting of," and preferably "consisting of," the SEQ ID NO:. As used herein, a "VHH family" or "family" refers to a group of VHH sequences that have the same length (i.e., they have the same number of amino acids in their sequence) and whose amino acid sequences share 80% or more (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more) sequence identity.

[0035] In one aspect, an equivalent sequence refers to amino acid substitutions that are preferably conservative amino acid substitutions (as defined herein); and / or the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to the amino acid sequence(s) identified herein; and / or refers to any amino acid sequence that has only three, two, or only one "amino acid difference(s)" (as defined herein) with one of the amino acid sequence(s) identified herein. More specifically, an equivalent sequence preferably has amino acid substitutions that are conservative amino acid substitutions (as defined herein); and / or the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to the amino acid sequence(s) identified herein.

[0036] The terms "epitope" and "antigenic determinant" may be used interchangeably and refer to a portion of a macromolecule, such as a polypeptide or protein, that is recognized by an antigen-binding molecule, such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain and / or a polypeptide of the invention, and more specifically by the antigen-binding site of said molecule. An epitope defines the minimal binding site for an immunoglobulin and thus represents the target of immunoglobulin specificity. An amino acid sequence (such as an immunoglobulin single variable domain, an antibody, a polypeptide of the invention, or a general antigen-binding protein or polypeptide or fragment thereof) that can "bind" or "specifically bind" to a certain epitope, antigen, or protein (or to at least a fragment thereof or epitope) is said to be "against" or "directed against" said epitope, antigen, or protein, or is a molecule that "binds" with respect to such epitope, antigen, or protein, or is said to be an "anti-" epitope, "anti-" antigen, or "anti-" protein (e.g., "anti-" APN).

[0037] Affinity describes the strength or stability of an intermolecular interaction. Affinity is expressed as K, which has units of moles / liter (or M). D Affinity is also commonly given by the binding constant, K A It can also be expressed as 1 / K D and (moles / liter) -1 (or M -1 ) for biological interactions that are considered meaningful (e.g., specific). D is typically 10 -12 M (0.001 nM) to 10 -5 M (10,000 nM). As the interaction becomes stronger, the K D will be lower.

[0038] The affinity of an intermolecular interaction between two molecules can be measured via various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al.

[12] ), in which one molecule is immobilized on a biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions, yielding k, koff measurements and therefore KD (or K) values. This can be performed, for example, using the well-known BIACORE® instrument (Pharmacia Biosensor AB, Uppsala, Sweden). The Kinetic Exclusion Assay (KINEXA®)

[13] is based on measuring binding events in solution without labeling the binding partners and kinetically excluding dissociation of the complex. Solution affinity analysis can also be performed using the GYROLAB® Immunoassay System

[14] , which provides a platform for automated bioanalysis and rapid sample turnover.

[0039] The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen-binding molecule or antigen-binding protein (such as an ISVD or polypeptide of the invention) can bind to. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. Typically, an antigen-binding protein (such as an ISVD and / or polypeptide of the invention) can bind to more than 10 different types of antigens or antigenic determinants. -5 From 10 -12 moles / liter or less, and preferably 10 -7 From 10 -12 The dissociation constant (K D ) will bind to their antigens. -4 Any K greater than moles / liter D The value (or 10 -4 Any K lower than liter / mol A values) are generally considered to indicate non-specific binding. Preferably, a monovalent ISVD of the invention will bind to a desired antigen with an affinity of about 10 μM or less, particularly about 5 μM or less, more particularly less than 1 μM, such as, for example, less than 500 nM, more particularly less than 200 nM, and even more particularly less than 10 nM, such as, for example, less than 500 pM, such as, for example, between 10 pM and 5 pM or less.

[0040] Specific binding of an antigen-binding protein, such as an ISVD, to an antigen or antigenic determinant may be determined in any suitable manner known to those skilled in the art, including, for example, saturation and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and various modifications of those known per se in the art; and other techniques described herein and preferably by flow cytometry or surface plasmon resonance.

[0041] In one embodiment, the binding of the polypeptide to porcine APN (pAPN) was determined via ELISA and flow cytometry on pAPN-expressing cells. However, because binding to immobilized antigen may not be equivalent to binding to cell membranes, the flow cytometry data was the most reliable. It demonstrated binding to the full-length protein in its native form expressed on biological membranes. Furthermore, we observed a clear correlation with binding in intestinal tissue, further confirming the flow cytometry data.

[0042] In one aspect, the present invention provides polypeptides that specifically bind to APN and also exhibit specific uptake by cells via the APN receptor, particularly by cells that express APN, and more particularly by enterocytes. As used herein, "enterocytes" or intestinal absorptive cells (also referred to as absorptive villous epithelial cells) are epithelial cells that line the inner surface of the small and large intestine and have phagocytic and transcytotic properties that transport intestinal pathogens or macromolecules across the epithelial barrier.

[0043] "Aminopeptidase N (APN)" (also known as CD13, ANPEP, PEPN, alanyl aminopeptidase) is a type II membrane glycoprotein, belongs to the family of membrane-bound metalloproteases, and is expressed in various tissues, such as the intestinal brush border membrane. Relevant structural information about APN may be found, for example, in UniProt or GenBank accession numbers, as depicted in Table 1 below. [Table 1]

[0044] As used herein, "APN" or "CD13" polypeptide refers to a protein encoded by a mammalian APN gene, and encompasses allelic variants containing conservative or non-conservative changes and biologically active fragments thereof, as well as artificial proteins that are substantially identical, i.e., at least 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to any one of the foregoing APN polypeptides. In specific embodiments, the APN polypeptide is at least 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to porcine or pig APN (Sus scrofa; accession number ADX53333.1). In a further embodiment, the APN polypeptides as defined herein are further characterized in that they are glycosylated.

[0045] By analogy, an "APN" or "CD13" polynucleotide is meant to encompass allelic variants and biologically active fragments containing conservative or non-conservative changes and any nucleic acid molecule substantially identical, i.e., at least 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to any one of the foregoing APN-encoding polynucleotides. In specific embodiments, the APN polynucleotide is at least 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid molecule encoding porcine APN (Genbank accession number HQ824547.1).

[0046] In one embodiment, the polypeptide of the present invention specifically binds to and is internalized by mammalian intestinal APN, particularly porcine APN, more particularly porcine intestinal APN, more particularly expressed on epithelial cells or enterocytes present in the small intestine, the APN comprising the amino acid sequence represented by protein accession number ADX53333.1. Unique data in the present invention demonstrate that after oral delivery, the polypeptide not only binds to APN, but also directs transcytosis (transport beyond the inside of the cell) across the epithelial barrier, with endocytosis (transport into the cell) by intestinal enterocytes and subsequent appropriate presentation to the mucosal immune system.

[0047] Cells expressing APN can be used to determine the uptake or internalization of the polypeptides of the present invention. "Expression" generally refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which mRNA is subsequently translated into a peptide, polypeptide, or protein. APN expression can be promoted or increased by methods involving the introduction of exogenous nucleic acid into cells. Such cells can contain a polynucleotide or vector in a manner that allows expression of the encoded APN polypeptide. A polynucleotide encoding APN as provided herein can be introduced into host cells as part of a circular plasmid containing an isolated protein-coding region, as linear DNA, or in a viral vector. Methods for introducing exogenous nucleic acid into host cells that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Host cell systems of the present invention include plant, invertebrate, and vertebrate cell systems.

[0048] Cells used to study internalization are primary cells isolated from the epithelial tissue of the small intestine, e.g., enterocyte preparations; or a range of recombinant cell lines expressing APN, including porcine intestinal epithelial cells (IPEC-J2, IPEC-I, IPI-2i), baby hamster kidney (BHK) cells (e.g., BHK21 cells), among others, or "mini-intestines" derived from either adult ISCs (enteroids / organoids) or induced pluripotent stem cells (iPSCs) (organoids). Additional cells may include, but are not limited to, insect cells, porcine kidney (PK) cells, porcine kidney cortex (SK-RST) cells, feline kidney (FK) cells, felis catus whole fetal cells (Fcwf-4), swine testis (ST) cells, African green monkey kidney cells (MA-104, MARC-145, VERO, and COS cells), Chinese hamster ovary (CHO) cells, human 293 cells, and mouse 3T3 fibroblasts, human colon carcinoma epithelial (CaCo2) cells, human lymphoblasts (Kasumi-3), human myeloblasts (Kasumi-4), human myeloblasts (Kasumi-6), human basophil cell line (KU812), human B lymphoblasts (SUP-B15), human epithelial kidney cortex cells (WT9-7, WT9-12). Insect host cell culture systems may also be used for expression of polypeptides in accordance with the present invention.

[0049] In this context, the selection of a suitable expression vector for expressing a polypeptide according to the present invention will naturally depend on the particular host cell used and will be within the skill of one of ordinary skill in the art. Examples of suitable expression vectors include pSport and pcDNA3 (Invitrogen), pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter and regulator sequences that may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus 2, polyoma virus, and simian virus 40 (SV40).

[0050] Alternatively, uptake or internalization of polypeptides can be determined by using porcine intestinal tissue in a gastrointestinal ligated loop experiment, as disclosed in the present examples. These data support the efficient transport of APN-targeted VHHs across the small intestinal epithelium.

[0051] Because APN sequences are known to exist in cells from various species, endogenous genes may be modified to permit or increase expression of APN polypeptides. Cells may be modified (e.g., by homologous recombination) to provide increased expression by replacing the naturally occurring APN promoter, in whole or in part, with all or part of a heterologous promoter, so that the cells express APN polypeptides at higher levels. The heterologous promoter is inserted in such a manner that it is operably linked to the endogenous APN-encoding sequence. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., the ada, dhfr, and multifunctional cad genes encoding carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and / or intron DNA may be inserted together with the heterologous promoter DNA. When linked to the APN-encoding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the APN-encoding sequence in the cell.

[0052] Alternatively, APN expression may also be induced by treatment with compounds known to induce APN expression in cells, such as treatment with basic fibroblast growth factor (bFGF)

[15] or with bestatin

[16] .

[0053] In one embodiment, an ISVD or polypeptide of the invention is said to be "cross-reactive" with respect to two different antigens or antigenic determinants (such as, for example, APN from different species of mammals, such as, for example, human APN, porcine APN, canine APN, feline APN, equine APN, bovine APN, rat APN, murine APN, and / or rhesus APN) if it is specific for those different antigens or antigenic determinants (as defined herein). It will be appreciated that an ISVD or polypeptide may be considered cross-reactive even though its binding affinity for two different antigens may differ, such as by a factor of 2, 5, 10, 50, 100, or even greater, provided that it is specific for these different antigens or antigenic determinants (as defined herein).

[0054] In a further aspect, the present invention relates to a polypeptide, which competes with a polypeptide as described herein, such as represented by SEQ ID NO: 6, 7 or 8, as determined by a competitive binding assay, such as, by way of example, flow cytometry, surface plasmon resonance or competitive ELISA, in particular competitive ELISA.

[0055] The present invention further relates to a method for determining a competitor, such as a polypeptide or small molecule, that competes with a polypeptide as described herein, such as one represented by any one of SEQ ID NOs: 6, 7 or 8, wherein the polypeptide as described herein competes with or cross-blocks a competitor, such as a polypeptide, for binding to an APN, such as porcine APN, and wherein the binding of the competitor to APN is reduced by at least 5%, for example, 10%, 20%, 30%, 40%, 50%, or more, for example, 80%, 90%, or even 100% (i.e., virtually undetectable in a given assay) in the presence of a polypeptide of the present invention, compared to the binding of the competitor to APN in the absence of the polypeptide of the present invention. In particular, a reduction of at least 80%, and preferably at least 90%, indicates competitive binding when measured by any one of the assays provided herein. Competition and cross-blocking may be determined by any means known in the art, such as, for example, flow cytometry, surface plasmon resonance, biolayer interference, or competitive binding assays such as competitive ELISA. In an aspect, the present invention relates to a polypeptide of the present invention, wherein the polypeptide cross-blocks the binding of a polypeptide represented by SEQ ID NO: 6, 7, or 8 to at least one APN. Assay conditions are applied as commonly known to those skilled in the art and / or as provided herein.

[0056] The terms "(cross-)blocking," "competitively binding," and "competing" are used interchangeably herein to refer to the ability of an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent to inhibit the binding of another immunoglobulin, antibody, ISVD, polypeptide, or binding agent to a given target. Methods for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent directed against a target (cross-)blocks competitively binds or is competitive as defined herein are described, for example, in Xiao-Chi Jia et al.

[17] or Miller et al.

[18] (incorporated by reference).

[0057] The present invention identifies a family of VHHs capable of binding to APN, inducing transport across the intestinal epithelium, and inducing an immune response. This family of single domain antibodies is characterized by the following complementarity determining regions (CDR1 to CDR3, respectively), as provided in Table 2. VHHs with such high sequence identity (80% or more identity), especially in CDR3, are predicted to recognize the same epitope and have the same functional characteristics. [Table 2]

[0058] In one aspect, the present invention provides a polypeptide comprising an immunoglobulin single variable domain (ISVD) that binds to intestinal aminopeptidase N, wherein the ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively): (i) CDR1 consists of the amino acid sequence represented by SEQ ID NO: 1, or a sequence having at least 70% identity thereto; (ii) CDR2 consists of the amino acid sequence represented by SEQ ID NO:3, or a sequence having at least 80% identity thereto; (iii) CDR3 consists of the amino acid sequence represented by SEQ ID NO: 4, or a sequence having at least 80% identity thereto.

[0059] In another aspect, the present invention provides a polypeptide comprising an immunoglobulin single variable domain (ISVD), which binds to intestinal aminopeptidase N, wherein the ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively); (i) CDR1 is SEQ ID NO: 1, and Amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NO: 1 Selected from the group consisting of; (ii) CDR2 is SEQ ID NO: 3, and Amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NO:3 Selected from the group consisting of; and (iii) CDR3 is SEQ ID NO: 4, and It is selected from the group consisting of amino acid sequences having 1, 2 or 3 amino acid difference(s) with SEQ ID NO:4.

[0060] In one embodiment, the variant amino acid is at position(s) 4, 6 and / or 7 of the amino acid sequence represented by SEQ ID NO: 1; and / or at position(s) 6, 10 and / or 12 of the amino acid sequence represented by SEQ ID NO: 4.

[0061] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to APN and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is: SEQ ID NO: 1; and - an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 1, wherein at position 4 I is changed to F, and / or where at position 6 N is changed to S, and / or where at position 7 H is changed to N, Selected from the group consisting of; (ii) CDR2 consists of SEQ ID NO: 3; and (iii) CDR3 is SEQ ID NO: 4; and an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 4, wherein at position 6 V is changed to A, and / or wherein at position 10 V is changed to L, and / or wherein at position 12 D is changed to E, is selected from the group consisting of:

[0062] In particular, the present invention provides a polypeptide comprising an immunoglobulin single variable domain (ISVD), wherein the ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 consists of SEQ ID NO: 1 or 2; (ii) CDR2 consists of SEQ ID NO: 3; and (iii) CDR3 consists of SEQ ID NO: 4 or 5.

[0063] Even more specifically, the present invention provides polypeptides comprising an immunoglobulin single variable domain (ISVD), wherein the ISVD comprises: - CDR1 is SEQ ID NO: 1, CDR2 is SEQ ID NO: 3, and CDR3 is SEQ ID NO: 4; or - CDR1 is SEQ ID NO: 2, CDR2 is SEQ ID NO: 3, and CDR3 is SEQ ID NO: 5, The CDR1, CDR2 and CDR3 combinations are selected from:

[0064] In a further embodiment, the present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD), said polypeptide comprising or consisting of the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or a sequence having at least 80% identity thereto, particularly at least 85% identity, more particularly at least 90% identity, even more particularly at least 92% identity thereto. In specific embodiments, the sequence variations in the CDRs are limited to position(s) 4, 6 and / or 7 of the amino acid sequence represented by SEQ ID NO: 1; and / or position(s) 6, 10 and / or 12 of the amino acid sequence represented by SEQ ID NO: 4. In another embodiment, there may be no sequence variations in the CDRs, and only the sequences of the FRs may differ, as characterized by SEQ ID NOs: 1 to 5, respectively. More specifically, the present invention provides a polypeptide comprising SEQ ID NO: 6 or a polypeptide having at least 80%, more particularly at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, and wherein CDR1 consists of SEQ ID NO: 1 or 2; CDR2 consists of SEQ ID NO: 3; and / or CDR3 consists of SEQ ID NO: 4 or 5.

[0065] In another aspect, the present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD), wherein the polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or a sequence having at least 70% identity, particularly at least 80% identity, more particularly at least 85% identity thereto.

[0066] In specific embodiments, sequence variation in the CDRs is limited to position(s) 4, 6, and / or 7 of the encoded amino acid sequence represented by SEQ ID NO: 1; and / or at position(s) 6, 10, and / or 12 of the amino acid sequence represented by SEQ ID NO: 4. In another embodiment, there may be no sequence variation in the amino acids encoding the CDRs, and only the nucleic acid sequences of the FRs may differ, as characterized by SEQ ID NOs: 1-5, respectively. More specifically, the present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD), said polypeptide comprising or consisting of the sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0067] It will be appreciated that the immunoglobulin single variable domains of the invention may be used, without limitation, as "building blocks" for the preparation of polypeptides that may optionally contain one or more additional immunoglobulin single variable domains that may serve as building blocks (i.e., directed against the same or another epitope on APN and / or against one or more other antigens, proteins or targets that are not APN). Thus, the polypeptides of the invention comprise at least one ISVD that binds to APN, and in particular that binds to APN and is endocytosed by cells that express APN. In general, a polypeptide or construct comprising or consisting essentially of a single building block, e.g., a single ISVD, will be referred to herein as a "monovalent" polypeptide and a "monovalent construct," respectively. A polypeptide or construct comprising two or more building blocks (e.g., ISVDs) will also be referred to herein as a "multivalent" polypeptide or construct, and the building blocks / ISVDs present in such a polypeptide or construct will also be referred to herein as being in a "multivalent format." For example, a "bivalent" polypeptide may comprise two ISVDs, optionally linked via a linker sequence, while a "trivalent" polypeptide may comprise three ISVDs, optionally linked via two linker sequences; and so forth.

[0068] Consequently, the ISVDs of the invention that bind to APN and are provided herein may be in essentially isolated form, or they may form part of a construct or polypeptide that may comprise or essentially consist of one or more ISVD(s) of the invention and may optionally further comprise, for example, one or more additional amino acid sequences encoding a protein or polypeptide, especially an antigenic protein or polypeptide (all optionally linked via one or more suitable linkers).

[0069] In a further embodiment, the present invention provides a "chimeric molecule" (optionally also referred to as a construct) comprising at least one polypeptide as defined herein, particularly at least one ISVD as provided herein, coupled, linked, or conjugated (directly or indirectly) to at least one compound having biological or functional activity, such as a chemical (e.g., a small molecule) or biological, particularly a drug / therapeutic agent, a bioactive compound, an antigen, a toxin, and / or a diagnostic agent (e.g., a label, marker, or imaging agent). In such an embodiment, the polypeptide or ISVD will act as a carrier for delivery of the compound across the intestinal barrier. The drug or therapeutic agent will be active in the intestinal submucosa or in the intestinal mucosa-associated lymphoid tissue, and / or the antigen will elicit an immune response in the intestinal submucosa or in the intestinal mucosa-associated lymphoid tissue. The therapeutic agent may be an anti-inflammatory agent, an anti-cancer agent, a cytotoxic agent, an anti-infective agent (e.g., antifungal, antibacterial, antiparasitic, antiviral, toxin, cytotoxic agent, radionuclide agent, etc.). Antigens include, but are not limited to, proteins, peptides, lipids, nucleic acids, glycolipids and glycoproteins, carbohydrates, oligosaccharides, and polysaccharides. Also, various drug delivery systems or (drug-loaded) carriers, e.g., based on nanoparticles (NPs), can also be conjugated to the polypeptides of the invention, including inorganic, magnetic, and polymeric particles or NPs.

[0070] The term "conjugated to," as used herein, refers, inter alia, to chemical and / or enzymatic conjugation resulting in a stable covalent linkage. Coupling to obtain a chimeric molecule can occur via specific amino acids (e.g., lysine, cysteine) present in the ISVD. As previously mentioned, any moiety, such as an agent (e.g., a protein, a nucleotide sequence, a lipid, a carbohydrate, a peptide, a drug moiety (e.g., a cytotoxic drug, an antibody-drug conjugate, or a payload), a tracer, or a detection agent), can be coupled with a specific biological or functional activity. In embodiments where the coupled moiety is a genetically encoded therapeutic or diagnostic protein or nucleotide sequence, the coupled moiety can be synthesized or expressed by either peptide synthesis or recombinant DNA methods, which are well known in the art. In another aspect where the conjugated moiety is a non-genetically encoded peptide, e.g., a drug moiety, the conjugated moiety can be artificially synthesized or purified from a natural source.

[0071] In further embodiments, the polypeptides or chimeric molecules as provided herein may further comprise one or more other groups, residues, moieties, or binding units. The one or more other groups, residues, moieties, or binding units are preferably selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc fragments or small proteins or peptides capable of binding to serum proteins, additional amino acid residues, tags, or other functional moieties, such as, for example, toxins, labels, radioactive chemicals, etc. The other groups, residues, moieties, or binding units may be, for example, chemical groups, residues, moieties, which may or may not themselves be biologically and / or pharmacologically active. For example, and without limitation, such groups may be linked to one or more of the ISVDs or polypeptides of the invention to provide "derivatives" of the polypeptides or constructs of the invention.

[0072] In general, various linkers known in the art can be used to link the ISVD of the present invention to the (biologically active) compound, agent, or moiety. As should be clear, both cleavable and non-cleavable linkers can be employed to achieve the desired release profile. In general, the optimal combination of linker and conjugation chemistry must be uniquely tailored to correlate with each unique aspect: the ISVD, the conjugated moiety, and the disease profile being treated. Still other suitable spacers or linkers will be clear to those of skill in the art and may be any linker or spacer commonly used in the art. In certain aspects, the linker or spacer is suitable for use in applications where pharmaceutical use is intended. For example, the linker between the lysine and the conjugate may also be, in certain aspects, a suitable amino acid sequence, and in particular, an amino acid sequence between 1 and 50 amino acids, or more specifically, an amino acid sequence between 1 and 30 amino acid residues. Some examples of such amino acid sequences include the Gly-Ser (GS) linker. Still other suitable linkers generally include organic compounds or polymers, particularly those suitable for use in polypeptides for pharmaceutical use. By way of illustration, poly(ethylene glycol) moieties have been used to link antibody domains. It is within the scope of the present invention that the length, degree of flexibility, and / or other characteristics of the linker may have some effect on the properties of the final ISVD conjugate of the present invention, including, but not limited to, affinity, specificity, or avidity for specific targeting. Based on the disclosure herein, one skilled in the art will be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a particular ISVD of the present invention.

[0073] The small size and rapid renal clearance of VHHs are sometimes undesirable characteristics for providing long-term protection in certain diseases. Several approaches ensure an extended half-life and are considered embodiments of the present invention. Thus, the efficacy of current VHHs can be improved by fine-tuning their residence time. The VHHs provided herein can be chemically modified to increase their molecular weight. Such chemical modification, for example with polyethylene glycol (PEG) groups, may also protect VHHs against proteases. Another strategy for extending the half-life of VHHs is by coupling VHHs to long-lived serum proteins or to building blocks that target these long-lived proteins. Furthermore, it has been observed that small, negatively charged proteins remain in the circulation longer than neutral proteins. There are several strategies for adding negative charges to proteins, including the addition of sialic acid polymers (polysialylation) or hydroxyethyl starch (HESylation), and by fusion with amino acid residues of the highly sialylated beta-carboxy-terminal peptide (CTP) found in the human chorionic gonadotropin (hCG) hormone. Finally, "Fc domain-based fusion constructs" are also widely used to extend the half-life of therapeutic proteins, including VHHs. Therefore, a further strategy is to fuse the VHHs of the present invention to the Fc region of an IgG molecule or other Fc moiety. However, other Fc domains, such as the Fc domain of IgA, which is more stable in the small intestinal environment, are also suitable.

[0074] For example, to increase affinity and avidity and at the same time allow for easy purification, the VHH domains of the present invention are fused to the Fc domain of mouse IgG, more specifically to mouse IgG2a, also referred to herein as VHH-MG fusions. VHHs of the present invention fused to the Fc domain of porcine IgG or porcine IgA are also part of the present invention and are referred to herein as VHH-PG or VHH-PA fusions, respectively. Suitable Fc domains or portions thereof are provided by way of example in Figure 10.

[0075] In one embodiment, the invention provides chimeric molecules comprising at least one polypeptide, particularly an ISVD as provided herein, and an Fc region or Fc binding portion, particularly an IgG or IgA Fc domain, such as, by way of example, an Fc domain comprising or consisting of any one of SEQ ID NOs: 18 to 21. In particular embodiments, the invention provides polypeptides comprising an ISVD comprising SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; fused to an Fc domain comprising or consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 or a portion thereof, or a sequence having at least 80%, at least 85%, or at least 90% identity thereto.

[0076] The polypeptides of the invention, in their broadest sense, and in particular the immunoglobulin single variable domains as described herein, are not limited to a particular biological source or to a particular method of preparation, including: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanizing" a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by "mutating" a naturally occurring VHH domain to reduce binding to pre-existing antibodies or by expression of a nucleic acid encoding such a mutated VHH domain; (5) by "camelizing" a naturally occurring VH domain from any animal species, and in particular from a mammalian species such as from humans, or by expression of a nucleic acid encoding such a camelized VHH domain. (6) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (7) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences, as known per se; (8) by preparing a nucleic acid encoding a Nanobody® using techniques for nucleic acid synthesis, as known per se, followed by expression of the nucleic acid; and / or (9) by any combination of one or more of the above.

[0077] The ISVD is "camelized" by replacing one or more amino acids in the amino acid sequence of a naturally occurring V domain from a conventional four-chain antibody with one or more amino acid residues occurring at the corresponding position(s) in the VH domain of a heavy-chain antibody. This can be performed by any method known to those skilled in the art. Such "camelizing" substitutions are preferably inserted at amino acid positions forming and / or occurring at the VH-VL interface and / or at the so-called camelid hallmark residues, which are well known to those skilled in the art and defined, for example, in WO 94 / 04678 and by Davies and Riechmann

[19] .

[0078] Furthermore, and as known to those skilled in the art, various production platforms, such as mammalian cells, yeast, plants, etc., can be used for the production of complex glycoproteins such as antibodies. Mammalian cell-based production, such as Chinese hamster ovary (CHO) cells, is straightforward for antibody production. Transgenic plant and yeast expression systems also show great potential. Yeasts, such as Komagataella phaffii

[20] , previously named Pichia pastoris and better known under older nomenclature, have distinct advantages over plant expression systems in terms of short production times, cost-effectiveness, ease of genetic manipulation, and scalability. Moreover, P. pastoris secretes extremely low amounts of endogenous proteins, and therefore, secreted recombinant proteins are highly enriched and concentrated, facilitating downstream processing. On the other hand, the glycosylation profile of recombinant proteins in plants is much more similar to that of animal cells than yeast expression platforms. In one aspect, the present invention provides for the production of polypeptides, particularly ISVDs, and even more particularly chimeric molecules as provided herein, using yeast, particularly Pichia pastoris.

[0079] Multivalent polypeptides or chimeric molecules of the invention can generally be prepared by a method comprising at least the steps of suitably linking an ISVD and / or monovalent polypeptide of the invention to one or more additional ISVDs or other compounds as described herein, optionally via one or more suitable linkers, so as to provide a polypeptide or chimeric molecule of the invention. Polypeptides of the invention can also generally be prepared by a method comprising at least the steps of providing a nucleic acid encoding a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such a method can be carried out in a manner known per se and that will be clear to the skilled artisan, for example, based on the methods and techniques further described herein.

[0080] In a further aspect, the present invention relates to a vector comprising a nucleotide sequence as provided herein. As used herein, the term "vector" encompasses any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus, AAV, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and appropriate DNA sequences necessary for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Typically, an "expression vector" comprises a nucleotide sequence in which an expressible promoter or regulatory nucleotide sequence is operably linked to or associated with a nucleotide sequence or DNA region encoding mRNA, such that the regulatory nucleotide sequence controls the transcription or expression of the associated nucleotide sequence. Typically, a regulatory nucleotide sequence or promoter of a vector is not operably linked to an associated nucleotide sequence as found in nature and is therefore heterologous to the coding sequence of the DNA region to which it is operably linked.

[0081] Another aspect relates to a host cell or expression host comprising a polypeptide, or chimeric molecule, or nucleic acid sequence provided by the present invention. The terms "host cell," "expression host," or "host" refer to a cell line used to express a protein or nucleic acid of interest in a recombinant manner (formed by genetic recombination). Although this is not always strictly required (e.g., in the case of plant cells, the plant itself can be used to produce the recombinant protein), the host cell can refer to a "higher eukaryotic cell" (e.g., a CHO cell line) and is typically part of a cell culture (e.g., a cell line such as an HEK or CHO cell line). Host cells can also refer to "lower eukaryotic cells" as used herein when referring to filamentous fungal cells or yeast cells. Yeast cells may be from the species Saccharomyces (e.g., Saccharomyces cerevisiae), Hansenula (e.g., Hansenula polymorpha), Arxula (e.g., Arxula adeninivorans), Yarrowia (e.g., Yarrowia lipolytica), Kluyveromyces (e.g., Kluyveromyces lactis), or Komagataella phaffii (Pichia pastoris) and may also be used herein. According to a particular embodiment, the lower eukaryotic cell is a Pichia cell, and in the most particular embodiment, a Pichia pastoris cell. "Prokaryotic" host cells typically refer to non-pathogenic prokaryotes, such as bacterial cells, e.g., E. coli, Lactococcus, and Bacillus species.

[0082] In a further aspect, the current invention provides an ISVD, polypeptide, chimeric molecule, nucleic acid, vector, host cell, or composition provided herein for use as a medicine, more particularly for use in preventing, treating, and / or reducing symptoms of intestinal diseases, and even more particularly for use in oral vaccination against intestinal diseases. As used herein, "intestinal diseases" refers to infections of the digestive tract or intestine and inflammatory diseases of the digestive tract or intestine, more particularly of the small intestine (e.g., inflammatory bowel disease, Crohn's disease, or ulcerative colitis). Such inflammation may be caused by, for example, an autoimmune disease. Infections by intestinal pathogens include infections by species selected from the group of genera consisting of pathogenic Escherichia coli (ETEC, EHEC, EPEC, STEC, ...), Vibrio, Campylobacter, Clostridium, Salmonella, Yersinia, Lawsonia, Rotavirus, Shigella, PEDV, Cryptosporidium, and Isospora. In particular, the present invention provides methods for treating and / or preventing intestinal diseases by administering to a subject a polypeptide, chimeric molecule or composition of the present invention. In one embodiment, administration is oral.

[0083] The ISVDs provided herein can function as carriers for targeting compounds, particularly bioactive compounds, and more particularly antigens, across the (gastro)intestinal mucosa. In one aspect, the present invention focuses on polypeptides or ISVDs as carriers for targeted vaccine delivery to the gastrointestinal epithelium, e.g., inducing a strong mucosal immune response against gastrointestinal pathogens. As used herein, "mucosal immune response" refers to an immune response (humoral and cellular) that occurs in the membranes of the intestinal, urogenital, and / or respiratory mucosa, i.e., surfaces that come into contact with the external environment. In the context of the present invention, a mucosal immune response is an intestinal mucosal immune response, particularly the production of immunoglobulin A (IgA), that occurs in the membranes of the mucosa of the small intestine. The present invention also provides methods for treating intestinal diseases and / or inducing an immune response against intestinal pathogens in a subject by orally administering to the subject an ISVD, polypeptide, chimeric molecule, or composition as provided herein.

[0084] In another aspect, the present invention relates to the use of an ISVD, polypeptide and / or chimeric molecule of the present invention in the preparation of a pharmaceutical composition. Thus, also provided are pharmaceutical compositions and their use in one or more of the methods of treatment described herein. Typically, pharmaceutical compositions comprise an ISVD, polypeptide and / or construct as described herein and a pharmaceutically acceptable carrier and / or excipient, optionally combined with an adjuvant.

[0085] A "carrier" or "adjuvant," particularly a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant," is any suitable excipient, diluent, carrier, and / or adjuvant that does not itself induce the production of antibodies harmful to the individual receiving the composition, nor does it evoke protection. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., that can be administered to an individual together with a compound without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably one that is relatively non-toxic and safe to patients at concentrations that are compatible with the effective activity of the active ingredient, such that any side effects attributable to the carrier do not impair the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen.

[0086] Suitable carriers or adjuvants typically include one or more of the following non-exhaustive list of compounds contained in large, slowly metabolized macromolecules: proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, stabilizers, flavorings, or colorings. "Diluents," particularly "pharmaceutically acceptable vehicles," include vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting agents, emulsifiers, pH buffers, and preservatives may also be included in such vehicles.

[0087] The polypeptides and chimeric molecules of the present invention, and optionally, pharmaceutically acceptable carriers and / or excipients, can be administered by any suitable route, including any commonly known to those skilled in the art. For treatment, the pharmaceutical compositions of the present invention can be administered to any patient according to standard techniques. Administration can be by any appropriate mode, including oral, parenteral, topical, nasal, ophthalmic, intrathecal, intracerebroventricular, sublingual, rectal, or vaginal, and is preferably oral. Further techniques, such as nanotechnology and formulation as aerosols or inhalants, are also within the scope of the present invention. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, coadministration with other drugs, contraindications, and other parameters to be considered by the clinician. The dosages and concentrations of carriers, excipients and stabilizers should be safe for subjects (humans, pigs, mice and other mammals) and include buffers such as phosphate, citrate and other organic acids; antioxidants such as vitamin C; proteins such as small polypeptides, serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as PVP; amino acids such as aminoacetate, glutamate, asparagine, arginine, lysine; glycose, disaccharides and other carbohydrates such as glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol, sorbitol; counterions such as Na+, and / or surfactants such as TWEEN®, PLURONICS® or PEG.

[0088] A further aspect of the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: - expression of a polypeptide or chimeric molecule as provided herein in a suitable expression system or host cell, in particular Pichia pastoris, and -purification or isolation of the APN-binding polypeptide The present invention relates to a method for producing the APN-binding polypeptide of the present invention, comprising: By purification or isolation of the expressed polypeptide is meant, by way of example and without limitation, affinity-based purification such as affinity chromatography, affinity purification, immunoprecipitation, protein detection, immunochemistry, surface-display, etc., all of which are well known in the art.

[0089] Yet another aspect of the present invention relates to a kit comprising a polypeptide, chimeric molecule, or nucleic acid according to the present invention. The kit may further comprise a combination of reagents such as buffers, molecular tags, vector constructs, reference sample materials, and suitable solid supports, cells, nucleic acids, etc. Such kits are useful for any of the present applications of the present invention as described herein. Also encompassed within the scope of the present invention is a solid support or resin comprising a polypeptide comprising an APN-binding ISVD according to the present invention. Non-existent examples of suitable solid supports include beads, columns, slides, chips, or plates. More specifically, the solid support may be in particulate form (e.g., beads or granules commonly used in extraction columns) or in sheet form (e.g., membranes or filters, glass or plastic slides, microtiter assay plates, dipsticks, capillary filling devices, or the like), which may be flat, pleated, or hollow fibers or tubes. Immobilization may be either non-covalent or covalent, using techniques known in the art.

[0090] The following examples are provided to illustrate methods, compositions, and results in accordance with the disclosed subject matter. These examples are not intended to encompass all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which would be apparent to one skilled in the art. [Example]

[0091] example material and method 1. Vectors, strains, and cell lines For VHH library preparation, we used the pMECS phage display vector system, which contains a pelB signal sequence for secretion of expressed VHHs into the bacterial periplasm and adds HA and His6 tags at the VHH carboxy terminus to enable purification. The yeast expression vector pPICZαH6E (NCBI accession number KM035419.1) (designated pKaiGG), modified for GoldenBraid cloning, was used to clone VHH-MG (MG: Fc domain of mouse IgG) fusions under the control of the methanol-inducible AOX1 promoter. The vector contains a Zeocin resistance marker for selection in bacteria and yeast cells. The E. coli strains TG1 and DH5α were used for the construction of the VHH library and fusion expression vectors, respectively. VHH-MG fusions were expressed in the P. pastoris wild-type strain NRRL Y-11430 (ATCC 76273). Stable APN-expressing cell lines (porcine intestinal epithelial cells IPEC-J2 and baby hamster kidney BHK-21) were obtained by transfection and incubated as previously described [3, 4].

[0092] 2. Purification of Intestinal APN Enterocytes were isolated from the small intestine of 3-week-old piglets according to the method of Lundqvist, Hammarstroem, Athlin, and Hammarstroem

[21] . After analysis using a light microscope, a purity of more than 85% of enterocytes was observed. Brush border membrane vesicles (BBMVs) were then isolated from the enterocytes as described by Melkebeek, Rasschaert, Bellot, Tilleman, Favoreel, Deforce, De Geest, Goddeeris, and Cox [3]. The resulting BBMVs were then lysed in 9 volumes of lysis buffer (100 mM Tris-HCl, 300 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM leupeptin, 5 mM DTT, 1 mM AEBSF), sonicated on ice for 2 minutes, rotated at 4°C for 30 minutes, and centrifuged at 17,400 g for 15 minutes at 4°C. The supernatant was collected and dialyzed against PBS using a 10 kDa MWCO dialysis cassette (Thermo Scientific). Protein concentration was determined by BCA reaction (Pierce BCA Protein Assay Kit) according to the manufacturer's recommendations. The lysate was stored at -20°C until further use.

[0093] Intestinal pAPN was purified from these lysates by immunoprecipitation. First, affinity-purified rabbit anti-APN IgG (in-house production) was crosslinked to Protein A Sepharose beads (CL-4B, Sigma). To this end, 50 mg of beads were washed three times with 1 ml of distilled water and centrifuged at 400 g for 2 min to remove salts. Rabbit anti-APN IgG (4 mg) was added to the beads in PBS for 1 h at room temperature (RT). Next, the beads were washed twice with 1 ml of 0.2 M sodium borate buffer (pH 8.0), followed by the addition of 1 ml of 0.2 M sodium borate (pH 9.0) + 20 mM dimethyl pimelimidate (DMP, Sigma) for 30 min at room temperature. After crosslinking, the beads were washed twice with 1 ml of 0.2 M ethanolamine + 50 mM ammonium bicarbonate buffer (pH 8.0) for 1 h. Next, the beads were washed three times with PBS to remove uncrosslinked IgG before adding elution buffer (0.2 M glycine-HCl, 50 mM L-arginine, 1 M NaCl (pH 2.0)). The beads were washed three times again in PBS and stored at 4°C until further use. Finally, the anti-APN beads were incubated with 1 ml of BBMV lysate on a rotating wheel overnight at 4°C. The beads were centrifuged (400 g, 2 min, 4°C), and the supernatant was collected. The beads were washed three times with PBS to remove unbound proteins. Next, APN was eluted from the beads with elution buffer for 10 min at RT. After centrifugation, the supernatant was collected and neutralized with 1 M Tris-HCl (pH 9.0). The eluted APN was immediately dialyzed in PBS and stored at -20°C. Concentration was determined by Pierce BCA Protein Assay Kit, and purity (>95%) was assessed by silver staining (Pierce® Silver Stain Kit; Thermo Scientific) performed according to the manufacturer's instructions.

[0094] 3. Generation of pAPN-specific VHH Library Porcine APN-specific VHHs were developed by the VIB Nanobody Core Facility in Brussels (Belgium) as previously described

[22] . Briefly, two llamas were subcutaneously immunized with 320 μg (per animal) APN isolated from pig kidney (Sigma, catalog no. L6007-50UN) on days 0, 7, 14, 21, and 28. On day 35, each llama received a final booster with 200 μg APN isolated from pig intestine. Porcine intestinal APN was purified as described above. Gerbu P (GERBU Biotechnik) was used as an adjuvant for all immunizations. On day 40, anticoagulated blood was collected for lymphocyte preparation. Total RNA from peripheral blood lymphocytes (PBLs) was used as a template for single-stranded cDNA synthesis with an oligo(dT) primer. Using this cDNA, VHH-encoding sequences were amplified by PCR, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phagemid vector pMECS, thus fused to a histidine tag. Electrocompetent E. coli TG1 cells were transformed with the recombinant pMECS vector. Independent VHH libraries were obtained from each llama, and each was subjected to panning (in solution) performed in BHK-21 cells stably expressing pAPN. The phage output of the first panning round from the two libraries was pooled, and the pool was used for further panning rounds. 190 randomly selected colonies (95 from each round) from the second and third panning rounds were sequenced and then grouped based on their CDR3 sequences. Using crude periplasmic extracts, 28 unique VHH (Nanobody®) sequences were analyzed by flow cytometry for specificity to pAPN using pAPN-expressing BHK-21 cells.

[0095] 4. Construction of VHH-MG fusion expression vector To clone the in silico-designed VHH-MG fusions into the yeast expression vector pKaiGG, customized reagents designed for Gibson Assembly® cloning, a synthetically produced DNA fragment encoding a VHH fused to the Fc tail of mouse IgG2a (Hinge-CH2-CH3; NCBI accession number KC295246.1), with a 40-base overlap between the insert and the vector, were obtained from SGI-DNA (La Jolla, California). The expression vector pKaiGG, adapted for Golden Gate cloning, was digested with SapI and column-purified (GeneJET PCR purification kit, ThermoFisher catalog number K0701). The VHH fragments were always assembled with the same Fc fragment and cloned in-frame at the 5' end with the α-factor secretion signal sequence (from a novel P. pastoris expression vector designated pPICZH6E; GenBank accession number KM035419) and at the 3' end with a transcription termination sequence into a proprietary SapI-digested pKaiGG vector via the BioXp™ 3200 system in an approximately 18-hour reaction and transformed into E. coli DH5α. Upon plasmid preparation and confirmation of the insert sequence, yeast cells were transformed with the PmeI-linearized vector, and positive transformants were selected in YPD (1% yeast extract, 2% peptone, 2% dextrose) supplemented with 50 mg / ml Zeocin (Invitrogen).

[0096] 5. Production of VHH-MG fusions in P. pastoris Expression and secretion of VHH-MG fusions in culture medium was first analyzed in 2 ml cultures. On day 1, 2 ml of BMMY medium (1% Bacto yeast extract, 2% peptone, 1.34% YNB, 0.1 M potassium phosphate (pH 6), 1% glycerol) containing 25 mg / ml Zeocin® was inoculated with individual transformants and incubated at 28°C for 48 hours with shaking. For each VHH-MG construct, four independent transformants were grown in 24-well plates. Cells were then pelleted at 1500 g for 10 minutes and resuspended in 2 ml of BMMY medium (1% Bacto yeast extract, 2% peptone, 1.34% YNB, 0.1 M potassium phosphate (pH 6), 1% methanol) to induce VHH-MG expression. Cultures were incubated with shaking at 28°C for 48 hours and spiked with 1% methanol (v / v) every 12 hours. After 48 hours of induction, yeast cells were pelleted and supernatants were analyzed by SDS-PAGE, Western blot and ELISA to assess the accumulation level of secreted VHH-MG fusions in the medium.

[0097] 6. Affinity Purification of VHH-MG Fusions Yeast transformants that produced high levels of VHH-MG were selected for upscaling to 1 L cultures using similar growth and induction conditions as described above for 2 ml cultures. P. pastoris cultures grown in baffled flasks were harvested 48 hours after induction. The culture supernatant was filtered using a 0.22 μM PES membrane filter (Millipore) and affinity-purified on a 1 ml HiTrap MabSelect SuRe Protein A column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 300 mM NaCl buffer, pH 7.8. Bound proteins were eluted with 1 M arginine, pH 2.7, and immediately neutralized with 1 M Tris, pH 9. Corresponding fractions containing high concentrations of VHH-MG were pooled and subjected to size-exclusion chromatography (Superdex 200, GE Healthcare). Fractions containing the major antibody peak in the chromatogram were pooled, concentrated by dialysis against 20% polyethylene glycol in PBS, and stored at −80° C. until use. Protein concentration was determined by OD280 measurement.

[0098] 7. SDS-PAGE and Western Blotting VHH-MG fusions (crude / purified) were analyzed under reducing conditions on a commercially available 4-20% gradient gel (Bio-Rad) and stained with Coomassie G-250 according to the manufacturer's instructions. For Western blotting, proteins were transferred to polyvinylidene difluoride membranes (PVDF) using a semi-dry transfer method (Bio-Rad). The blotted membranes were blocked overnight at 4°C with 2% nonfat milk in PBST (PBS + 0.1% (v / v) Tween-20). The membranes were then probed with anti-mouse IgG (GE Healthcare, NXA931) conjugated to horseradish peroxidase (HRP) diluted 1:2000 in blocking solution and incubated for 1 hour at RT. The membranes were then washed three times with PBST, and bands were visualized by adding HRP substrate (WesternBright ECL, Advansta). Membranes were imaged using a ChemiDoc MP imaging system (Bio-Rad) and analyzed by ImageLab software (Bio-Rad).

[0099] 8. Antigen binding ELISA 96-well ELISA plates (Nunc Polysorp®) were coated with 400 ng of APN (Sigma) diluted in PBS for 2 h at 37°C. The plates were then washed three times with PBST and blocked overnight at 4°C with 3% (w / v) gelatin prepared in PBS and 0.05% Tween-80. Following three washes with PBST, VHH-MG fusions were serially diluted in dilution buffer (2% nonfat milk + PBS + 0.05% Tween-20), added to each well, and incubated for 1 h at RT. An in-house produced pAPN-specific mAb (IMM013, see [4]) was used as a positive control, while a GFP-specific VHH fused to mouse IgG3 (GBP-MG) was used as a negative control

[23] . After washing, bound VHH-MG fusions were detected with anti-mouse IgG (GE healthcare, NXA931) diluted 1:5000 in dilution buffer. Following 1 h of incubation at RT, plates were washed three times with PBST and revealed by adding HRP substrate (one SIGMAFAST™ OPD tablet dissolved in 20 ml deionized water). Finally, the reaction was stopped with 1 M hydrochloric acid, and the optical density of the colorimetric reaction was measured at 492 nm (VersaMax™).

[0100] 9. Flow Cytometry The binding of various VHH-MG fusions to APN-expressing cell lines was analyzed using flow cytometry. Wild-type and APN-transfected IPEC-J2 and BHK-21 cell lines were grown to 90% confluence and detached with StemPro Accutase (Gibco). Detached cells (3.0 x 10 5) were transferred to 200 μl of culture medium in a conical-bottom 96-well microtiter plate (Gibco) and centrifuged at 350 g and 4°C for 3 minutes. Cells were incubated with 2 μg / ml of VHH-MG fusion or anti-APN mAb (clone IMM013) on ice for 30 minutes. Detection of bound VHH-MG fusion was performed with AF647-conjugated anti-mouse IgG2a antibody (4 μg / ml; Invitrogen, A21241) for IMM013 and with AF647-conjugated anti-mouse IgG1 antibody (4 μg / ml; Invitrogen, A-21240) for IMM013. Cells were incubated on ice for 30 minutes. Dead cells were excluded using Sytox blue staining (5 nM; Molecular probes). For each condition, a total of 10,000 viable single cells were measured (Cytoflex, Beckman Coulter).

[0101] 10. VHH-MG Endocytosis Assay APN-expressing BHK-21 cells were seeded on top of sterile coverslips in 24-well plates (1.0x10 in 1 ml). 5Cells) and cultured until a monolayer formed. Cells were washed twice with ice-cold PBS and stored on ice before adding VHH-MG fusions or IMM013 (250 μl; 100 μg / ml) to the cells in ice-cold culture medium. After 30 or 60 minutes of incubation at 4°C, cells were washed three times with ice-cold PBS and incubated at 37°C, 5% CO2, in warm culture medium for 30 minutes. After washing twice in PBS, cells were fixed with 500 μl of 4% paraformaldehyde for 10 minutes at room temperature (RT). The presence of VHH-MG or IMM013 on the cell membrane was then detected with AF568-conjugated anti-mouse IgG (H+L) (2 μg / ml; Invitrogen, A-11004) for 30 minutes at RT and protected from light. After washing three times with PBS + 1% FCS, the cells were permeabilized with 250 μl of 0.2% Triton-X100 for 2 minutes and washed again with PBS + 1% FCS. Both VHH-MG and IMM013 were then detected using FITC-conjugated anti-mouse IgG (1 / 50; whole molecule) (Sigma, F2883) for 30 minutes at room temperature and protected from light. Nuclei were visualized by Hoechst staining (1 / 100 dilution). After three washes, the coverslips were removed and mounted on microscope slides in mounting solution (anti-fade agent 1,4-diazobicyclo-2,2,2-octane-DABCO™). Images were taken using a confocal microscope (Leica).

[0102] 11. In vivo uptake of anti-APN VHH-MG Three female, 5-week-old piglets were used to evaluate the uptake of VHH-MG in ligated gastrointestinal loops as previously described

[24] . Briefly, after anesthesia and laparotomy, the jejunum was located and six 3-cm loops were created with 10-cm intervals between each loop, avoiding the Peyer's patches. Blood supply was secured by placing ligatures between the mesenteric arcades. A pAPN-specific mAb (IMM013, see [4]) was used as a positive control, while GBP-MG was used as a negative control

[24] . Anti-APN mAb IMM013 (1 mg) or equimolar amounts of different VHH-MG were diluted in 3 ml of PBS and injected into the lumen of the loop. Upon injection, each loop was returned to the abdominal cavity, and the cavity was closed. After 5 hours of incubation, the animals were euthanized by an overdose of sodium pentobarbital, and tissue samples were collected from the intestine, loops, and mesenteric lymph nodes. The tissue samples were embedded in 2% Methocel® MC (Fluka), flash-frozen in liquid nitrogen, and stored at −80°C until use. All animal procedures were approved by the Ethics Committee of the Faculty of Veterinary Medicine, Ghent University (EC 2018-04).

[0103] 12. Immunohistochemistry Cryosections (8 μm) of porcine ileum obtained from 3-week-old piglets or digestive loops were cut using a Leica CM3050 S cryostat, mounted on APES-coated slides, dried (30 min, 40°C), and fixed in acetone for 10 min at -20°C. Subsequently, the cryosections were incubated in ammonium chloride buffer (50 mM, pH 8) for 30 min and thoroughly washed. All washing steps were performed in PBS at RT. Fc receptors were blocked with PBS containing 10% sheep or goat serum for 30 min at 37°C. Binding of VHH-MG or IMM013 was detected by incubation with FITC-conjugated sheep anti-mouse IgG (10 μg / ml; Sigma; F2883) or goat anti-mouse IgG2a for 1 h at 37°C. Nuclei were counterstained with Hoechst (10 μg / ml). To assess endocytosis, sections were stained with rabbit anti-pan-cytokeratin antibody (Abcam, ab9377) and detected with Texas Red-conjugated anti-rabbit IgG. Sections were washed in ultrapure water and mounted in mounting solution (DABCO). Tissues were imaged using a Leica DC 100 fluorescence microscope equipped with a Scion Corporation camera or a Leica confocal microscope. Images were analyzed and processed using Fiji.

[0104] 13. Oral immunization experiments All animal procedures were approved by the Veterinary Faculty Ethics Committee (EC2018-51). Twelve conventionally raised piglets (Belgian Landrace × Pietrain) from a Belgian farm were weaned at 3 weeks of age and transported to our facility. The animals were treated with colistin sulfate (Promycine®, 100,000 UI / kg animal body weight) for 5 days prior to the start of the experiment. The design of the oral immunization experiment is depicted in Figure 6. Animals were randomly divided into three groups: a mouse IgG2a control group and two groups receiving either the APN-specific VHH-MG 2L65 or 3L94. Animals were orally immunized for three consecutive days, followed by a booster immunization on day 14 after the primary immunization. Omeprazole (20 mg / animal) was administered 24 hours before each immunization to block HCl production in the stomach, and the animals were food-deprived 12 hours before immunization. Animals were orally immunized with 1 mg of either 3L94-MG or 2L65-MG, or an equimolar amount of irrelevant mouse IgG2a (Bio X cell; West Lebanon, USA) adjuvanted with an equal volume of 50 μg of cholera toxin (Merck, ref. C8052). Blood was collected on days 0, 9, 14, 21, and 28 post-primary immunization (ppi) to analyze the elicited immune response. On day 28 ppi, animals were euthanized by intravenous injection of 20% sodium pentobarbital (60 mg / 2.5 kg; Kela), and after exsanguination, small intestinal tissue samples were collected.

[0105] 14. Mouse IgG2a ELISA Blood was collected from the jugular vein into tubes containing gel and a coagulant accelerator (Vacutest, Kima). After incubation at room temperature for 1 hour, the tubes were centrifuged, and the serum was collected and inactivated at 56°C for 30 minutes. Serum samples were stored at -20°C until use. In-house produced mouse IgG2a monoclonal antibody was coated onto 96-well microtiter plates (Polysoap; Life Technologies) at 6 μg / ml in PBS for 2 h at 37°C. After overnight blocking in PBS supplemented with 0.2% Tween 80 and 3% BSA, diluted serum samples (starting dilution 1:10 in dilution buffer) were added to the wells. After incubation at 37°C for 1 h, the plates were washed and incubated with HRP-conjugated mouse anti-pig IgG (MT424, 1 / 1000; MabTech, Nacka Strand, Sweden) or HRP-conjugated goat anti-pig IgA (1 / 10,000; Bethyl; Montgomery, Texas, USA) for 1 h at 37°C. Following three washing steps, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was added, and the optical density was measured at 405 nm using a spectrophotometer (Tecan SpectraFluor) after 45 min of incubation at 37°C.

[0106] 15.ELIspot Blood was collected from the piglet's jugular vein onto heparin, and PBMCs were isolated by density gradient centrifugation using Lymphoprep® (Alere Technologies, Oslo, Norway). Red blood cells were lysed in ammonium chloride solution. The resulting PBMC fraction was washed twice in ice-cold PBS + 1 mM EDTA and resuspended at 1 x 107 cells / ml in CTL-Test BTM (Cellular Technology Limited, Cleveland, USA) supplemented with 1% penicillin (100 IU / mL), streptomycin (100 μg / mL), and kanamycin. Mononuclear cells (MCs) were isolated from mesenteric lymph nodes (MLNs), jejunal Peyer's patches (JPPs), jejunal lamina propria (JLPs), ileal Peyer's patches (IPPs), and ileal lamina propria (ILPs) and processed as previously described

[25] . Isolated MCs were resuspended in leukocyte medium (RPMI-1640 (Gibco) containing 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM l -glutamine, penicillin (100 IU / mL), streptomycin (100 μg / mL), and nonessential amino acids (1%)) and counted.

[0107] Multiscreen filter plates (96-well format, MAIPA4510, Millipore) were activated with 70% ethanol, washed with ultrapure water, and coated with 10 μg / ml mouse IgG2a overnight at 4°C. After washing, the plates were incubated in CTL-Test B medium at 37°C for 2 hours. Mononuclear cells from each tissue (2.5 × 105 / well) were added to the wells and incubated at 37°C for 18 hours in a humidified 5% CO2 atmosphere. The cells were then removed by extensive washing with PBS containing 0.1% Tween 20. After washing, HRP-conjugated anti-pig IgG (MT424, 1 / 1000; MabTech) or IgA (1 / 10,000; Bethyl Laboratories) was added in assay buffer (PBS containing 0.1% Tween 20 and 0.1% BSA) and incubated at room temperature for 1 hour. Finally, 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma) for membranes was added to the wells after three washing steps. The reaction was stopped by extensive washing with UP water, and the plates were dried overnight at 4°C. Images were taken using an immunospot reader (Luminoskan). Spots were manually counted based on size discrimination.

[0108] 16. Competition Assay Flow cytometry A flow cytometry-based competition assay was used to determine whether VHHs recognize similar epitopes on APN. APN-transfected BHK-21 cell lines were grown to 90% confluence and detached with StemPro Accutase (Gibco). Detached cells (3.0 x 10) were cultured at 4°C for 1 hour. 5) were transferred to a conical-bottom 96-well microtiter plate (Gibco) in 200 μl of culture medium and centrifuged at 350 g for 3 minutes at 4°C. Next, BHK-APN cells were first incubated with 2L65, 3L2, 3L73 VHH-MG or a negative control at 40 μg / ml on ice for 30 minutes. After washing with PBS + 1 mM EDTA, the cells were incubated with secondary labeled 2L65, 3L2, 3L73 VHH-MG (AlexaFluor405 Mouse IgG2a Zenon Labeling Kit) at 2 μg / ml on ice for 30 minutes. After washing with PBS + 1 mM EDTA, the cells were analyzed by flow cytometer (Cytoflex, Beckman Coulter). Dead cells were excluded using propidium iodide staining. A total of 10,000 viable, single cells were measured for each condition (Cytoflex, Beckman Coulter).

[0109] Biolayer Interference Epitope binning measurements were performed at room temperature using biolayer interferometry (BLI; Octet RED96). Here, 10 μg / ml biotinylated porcine APN (Sigma) in PBS was bound to a high-precision streptavidin (SAX) biosensor immersed in PBS, followed by the addition of VHH at 500 nM in PBS + 0.2% Tween-20 + 1% BSA (PBST + BSA). 2L65 VHH was then added at 250 nM in PBST + BSA. Data were analyzed using high-throughput epitope binning software.

[0110] ELISA 96-well ELISA plates (Nunc Polysorp®) are coated with 400 ng of APN (Sigma) diluted in PBS for 2 hours at 37°C. The plates are then washed three times with PBST and blocked overnight at 4°C with 3% (w / v) gelatin prepared in PBS and 0.05% Tween-80. After washing three times with PBST, VHH-MG fusions are added to each well at saturating concentrations in dilution buffer (2% nonfat milk + PBS + 0.05% Tween-20) and incubated for 1 hour at RT. After washing, biotinylated 2L65 VHHs are added to each well at 10 μg / ml in dilution buffer and incubated for 1 hour at RT. Bound biotinylated 2L65 VHHs are detected with streptavidin-HRP (ThermoFisher scientific) diluted 1:5000 in dilution buffer. Following a 1-hour incubation at RT, the plates are washed three times with PBST and revealed by adding HRP substrate (one SIGMAFAST™ OPD tablet dissolved in 20 ml of deionized water). Finally, the reaction is stopped with 1 M hydrochloric acid, and the optical density of the colorimetric reaction is measured at 492 nm (VersaMax™).

[0111] 17. Statistical analysis Data were analyzed using GraphPad Prism 6. Serum antibody levels and the number of circulating antigen-specific antibody-secreting cells were analyzed by two-way ANOVA and Dunnett's test for multiple comparisons. The number of intestinal antigen-specific antibody-secreting cells was analyzed by t-test and Holm-Sidak for multiple comparisons. The significance level was set at 0.05.

[0112] result 1. Construction of an APN-specific VHH library To prepare the anti-APNVHH library, two llamas were immunized five times with commercially available kidney pAPN. A sixth booster was given with intestinal pAPN encoded by the same gene and prepared by the procedure described in the Materials and Methods section. Two independent VHH phage display libraries were constructed, each containing approximately 10 8 The total number of VHHs consisted of 10 transformants. After two consecutive rounds of panning performed in stably transfected BHK21 cells expressing porcine aminopeptidase N, 190 clones were randomly selected and sequenced. Through comparative alignment, VHHs (B cell lineages) with a percent sequence identity of more than 80% in their CDR3 regions were grouped into one family because they most likely recognize the same epitope (see

[26] ), but their characteristics (e.g., affinity, expression level, stability, etc.) may differ. Thus, we obtained 28 unique VHHs belonging to 14 different families as potential binders to APN (Figure 4). These 28 unique VHHs were produced in E. coli cells, and crude periplasmic extracts were analyzed for their binding specificity via flow cytometry in BHK21 cells expressing pAPN. Parental, non-transfected BHK-21 cells served as negative control cells. An unrelated VHH (BCII10, specific for bacterial β-lactamase, described by Conrath et al.

[27] ) and mouse anti-APN mAb IMM013 [4] were used as negative and positive controls, respectively. This FACS analysis confirmed that 22 of the original 28 screened diverse VHHs belonged to 11 diverse families specific for porcine APN, demonstrating that panning of the library in pAPN-transfected cells highly enriched for APN-binding VHH clones (Figure 4).

[0113] 2. Construction of VHH-MG expression vector via BioXP DNA printer Because FACS analysis of panning in APN-transfected BHK-21 cells was performed with crude periplasmic extracts, the binding quality of the various VHHs had to be further analyzed with purified proteins to allow comparison between the various VHHs and to eliminate experimental variables such as nanobody expression, periplasmic extraction efficiency, etc. in the FACS analysis (Figure 4). It is important to clearly understand that because the VHH library with a C-terminal histidine tag was prepared in the suppressor E. coli TG1 strain, which reads the amber stop codon (TAG) as glutamine (Q), some VHHs may contain an amber stop codon within the VHH sequence. Thus, when the amber codon was identified by sequencing, it was also replaced with a glutamine codon.

[0114] Classically, selected VHHs are subcloned in direct fusion with affinity tags in nonsuppressor E. coli strains, but here we followed a new strategy. We subcloned VHHs in direct fusion with Fc fragments and produced the fusions in Pichia pastoris culture medium, offering the following advantages: the Fc could be used as an affinity tag for protein purification, it made VHH-Fc fusions bivalent, providing avidity effects, and it allowed comparison with the mouse monoclonal antibody IMM013 for binding to APN as a positive control. Twenty-eight selected VHHs were fused to the Fc domain of mouse IgG2a and cloned into an in-house P. pastoris expression vector. Two unrelated VHHs, V2

[28] and D3

[29] , specific for the F4 and F18 fimbriae of enterotoxigenic E. coli, respectively, were included as negative controls.

[0115] The sequences of the VHH and Fc domains were synthesized using the BioXP™ 3200 System and transformed into yeast expression vectors (Figure 1a). All 30 constructs successfully assembled by the BioXP DNA printer (along with 28 selected VHHs and two negative controls) were transformed into E. coli, and four colonies per construct were analyzed by colony PCR or restriction digestion followed by sequencing to identify clones with the correct insert. Twenty-three of the 30 constructs (77%) resulted in error-free clones by sequencing only one colony, whereas for seven VHH-MG constructs, two colonies had to be analyzed to obtain the correct clone; for only one clone, 2L58MG (3%), the sequence was incorrect in all four colonies, with one mutation in the coding sequence (Figure 1b). Coincidentally, this incorrect clone from family 12 also returned negative results in panning during VHH library preparation (Figure 4). Therefore, we omitted it from further analysis.

[0116] 3. Yeast-produced VHH-MG fusions efficiently bind to full-length, surface-expressed APN The 29 resulting VHH-MG-encoding constructs were then transformed into Pichia pastoris, and for each construct, four individual yeast colonies were screened to identify the transformant producing the highest amount of VHH-MG, which accumulated in the culture medium. Therefore, equal amounts of the supernatant were analyzed by SDS-PAGE and Western blotting to examine the signal intensity and protein integrity of the recombinant protein (Figure 5). Only for the best-expressing constructs, bands could be observed after SDS-PAGE; however, for most constructs, bands were clearly present at the expected molecular weight after Western blot analysis. The concentrations of the various VHH-MGs produced in P. pastoris were determined using purified V2-MG and D3-MG reference proteins and ranged from 4 to 60 mg / L in 2 ml of culture, based on analysis by ImageLab software. Only for some fusions, such as 3L11MG (family 10), 2L22MG (family 5) and V2-MG, degradation products were detected in addition to the full-length product, suggesting that the VHH has an effect on the stability of the VHH-MG fusions, as was also observed for the VHH-IgA fusions

[30] .

[0117] Next, we determined the binding specificity of the VHH-MG fusions in an ELISA specific for kidney APN (Fig. 2a). Three-fold dilutions of VHH-MG in culture supernatants were probed with anti-mouse IgG. The three strongest VHH-MGs, namely 2L48MG, 3L94MG, and 2L76MG, displayed strong binding activity to immobilized APN and belong to different CDR3 groups. These three VHH-MGs also showed good binding in a FACS assay performed after panning to screen for phage-displayed VHHs (Fig. 4). Interestingly, however, some families of VHH-MG fusions, such as family 4 (2L63-MG and 2L69-MG), were completely negative in ELISA, whereas they were strong binders in the FACS assay (Fig. 4). It is important to note that the relative binding activity on APN-coated wells in the ELISA assay did not correlate with the affinity of the VHH-MG fusions for APN expressed on cells. Of note, VHH screening was performed via FACS using cells expressing APN on their surface, and therefore binding to immobilized APN may differ from binding to cells, presumably because the epitope becomes less accessible during immobilization. Furthermore, because the experiments were performed with crude medium samples in which VHH-MG protein accumulation was not equilibrated, the ELISA signal may be related to protein concentration in the culture medium rather than affinity (Fig. 2a, Fig. 4).

[0118] The VHH-MG fusions were further evaluated for binding specificity and affinity in the parental cell line versus its derived transfected APN-expressing cell lines (BHK21 and IPEC-J2) via flow cytometry with equal amounts of VHH-MG protein (Figure 2b). Note that IPEC-J2 is a porcine small intestinal epithelial cell line that expresses low levels of APN on its surface, and therefore, only stably transfected cells were used. Based on the relative VHH-MG concentrations as determined by Western blotting, equimolar amounts of VHH-MG protein and a positive control (IMM013) were spiked into spent medium of P. pastoris. Several strong APN binders were identified through flow cytometry based on the number of cells that bound VHH-MG; in contrast, no binding was observed in the spent medium of untransfected BHK21 / IPEC-J2 cells. Interestingly, family 4 (2L63 and 2L69), which were negative in the APN-specific ELISA, contained strong binders in the cell-based binding assay (Figure 2b, Figure 7). These findings were consistent with the FACS data obtained from crude periplasmic extracts during VHH library screening (Figure 4). We found that the amplitude of binding activity in IPEC-J2-APN cells was significantly lower than that in BHK21-APN, which may be related to the amount of APN expressed on the IPEC-J2 cell membrane.

[0119] We selected at least one clone from each family that showed binding activity in flow cytometry screening (excluding families 6, 10, 11, and 14; families 6, 11, and 14 did not contain APN-binding VHHs; family 10 (3L11) displayed only degradation and low binding strength) and further evaluated their affinity to small intestinal tissue via immunohistochemistry (IHC). Based on their binding activity, several clones were selected and purified for in vivo analysis. Clones with low yields, such as 2L76-MG, 2L34-MG, and 2L52, were omitted from further analysis. Therefore, only six families containing strong binders (1, 4, 8, and 9; approximately 100,000 MFI) and moderate binders (5 and 12; greater than 200,000 MFI) were selected for upscaling. Each VHH-MG displayed binding to the intestinal ileum, whereas all controls were negative and showed no background; moreover, the intensity of the signal correlated with the binding activity in FACS analysis (FIG. 2b).

[0120] 4. Purified APN-binding VHH-MG fusions are internalized by intestinal epithelial cells For each family, a single VHH-MG fusion that displayed high expression and secretion in spent Pichia medium and strong binding by FACS was selected for upscaling. Secreted VHH-MG fusions were affinity purified on protein A and further evaluated by size-exclusion chromatography (SEC) (Figure 3a). For most fusions, the SEC profile showed two major peaks: one representing aggregates and multimers, and one representing monomers of the corresponding molecular weights of the two related VHH-MG polypeptides. The major fraction of VHH-MG in 2L76 was found in aggregates; therefore, we excluded it from further analysis (data not shown).

[0121] For the remaining six fusions, fractions corresponding to monomeric VHH-MG were pooled and further analyzed by SDS-PAGE under both reducing and non-reducing conditions to verify dimerization. Because the peaks for monomer and aggregates in the SEC profiles overlap slightly, the pooled purified VHH-MG still contains a small amount of aggregates. The non-reducing gel sample clearly showed an 80 kDa band (Figure 3b), which corresponds to monomeric VHH-MG composed of two 40 kDa VHH-MG polypeptides, as seen in SDS-PAGE under reducing conditions. Additionally, several higher molecular weight bands were observed, most likely representing aggregates or glycan variants.

[0122] Next, we tested the binding characteristics of various purified VHH-MG fusions in ELISA and FACS (Fig. 5a and 5b, respectively). A GFP-specific VHH fused to mouse IgG3 (GBP-MG) was used as a negative control

[23] . Although all six selected fusions showed strong binding to APN-expressing cells, the binding efficiency on immobilized antigen in ELISA varied greatly, as observed with crude samples (Fig. 4).

[0123] Because efficient endocytosis by target cell populations of potential vaccine delivery systems is highly important for achieving a strong immune response, we further investigated whether APN-binding VHH-MG fusions exhibit specific cellular uptake via the APN receptor. We used APN-expressing BHK21 cells due to their strong binding properties in FACS (Figure 5b). VHH-MG proteins were stained with different fluorescent molecules to distinguish membrane-associated VHH-MG from VHH-MG internalized via the APN receptor. Confocal imaging analysis clearly demonstrated APN-dependent uptake of VHH-MG fusions, as no independent uptake of VHH-MG fusions could be observed. Three VHH-MG fusions (3L94-MG, 2L65-MG, and 2L48-MG) were found to be the strongest APN binders and displayed clear uptake signals only in APN-expressing cells. As shown in Figure 11, endocytosis assays further confirmed that 3L2 and 3L73 VHH triggered internalization similarly to 2L65 VHH.

[0124] 5. In vivo behavior of selected APN-specific VHH-Fc candidates in the gastrointestinal ligated loop assay To evaluate the in vivo behavior of selected VHH-Fc IgG constructs, we performed a gastrointestinal ligated loop assay in the piglet small intestine (jejunum). 3L94-MG and 2L65-MG were able to bind to the entire apical epithelium of the villus. In contrast, 2L48 and 2L22 showed a more patchy binding profile. Similar binding profiles were observed across different animal models. In addition, 3L94-MG and 2L65-MG were endocytosed by enterocytes in a similar manner as IMM013. Based on this in vivo behavior in the gastrointestinal ligated loop assay, we concluded that 3L94-MG and 2L65-MG were the most suitable candidates for targeting antigens to the small intestine. The characterization of the CDR1-3 regions of these single-domain antibodies is provided in Table 3. Table 3 [Table 3]

[0125] 6. Oral immunization with APN-specific VHH-Fc elicits circulatory and small intestinal immune responses To further demonstrate the potential of the selected VHH-MG as a vaccine delivery system, oral immunization experiments in piglets were performed, and the subsequent systemic and small intestinal immune responses were assessed through the detection of mouse Fc IgG2a-specific serum antibody responses and antibody-secreting cells via ELISA and ELIspot, respectively. Figure 6b shows that upon oral administration of 2L65-MG, serum levels of mouse Fc IgG2a-specific IgG and IgA significantly increased compared with the control group. Surprisingly, 3L94 did not promote mouse Fc IgG2a-specific serum antibody responses. In addition, oral immunization with 2L65-MG significantly increased the amount of circulating mouse Fc IgG2a-specific IgA-secreting cells on day 21 after primary immunization compared with day 0 and the control group (Figure 6c). These results indicate that APN-specific VHH-MG promotes systemic immunity upon oral administration to piglets. To further demonstrate that the intestinal immune response was also enhanced, we estimated the presence of mouse Fc IgG2a-specific antibody-secreting cells in various small intestinal tissues via ELIspot. As shown in Figure 6d, 2L65-MG alone significantly enhanced the number of mouse Fc IgG2a-specific IgA-secreting cells in the mesenteric lymph nodes and ileal lamina propria and Peyer's patches at 28 dppi. Collectively, these results indicate that oral administration of 2L65-MG triggers both systemic and small intestinal immune responses in piglets.

[0126] 7. Competition Assay This flow cytometry-based competition assay was used to determine whether VHHs recognize similar epitopes on APN. As shown in Table 4, each VHH blocked the binding of the other VHH to BHK-APN cells, indicating that these VHHs are competing VHHs that bind to similar epitopes. Table 4: Flow cytometry-based competition assay. Data are expressed as median fluorescence intensity after subtraction of control values. [Table 4] In conclusion, the present invention provides a family of VHHs that share high sequence similarity (2L65, 3L2, 3L73) and that not only bind to APN-expressing cells but also trigger their transport across the small intestinal epithelium, and that have the unique feature of being able to induce systemic and intestinal IgA responses after oral administration, and in particular to induce antigen-specific IgA+ B cells.

[0127] literature [1] V. Snoeck, W. Van den Broeck, V. De Colvenaer, F. Verdonck, B. Goddeeris, E. Cox, Transcytosis of F4 fimbriae by villous and dome epithelia in F4-receptor positive pigs supports importance of receptor dependent endocytosis in oral immunization strategies, Vet. Immunol. Immunopathol., 124 (2008) 29-40. [2] B. Devriendt, BG De Geest, BM Goddeeris, E. Cox, Crossing the barrier: Targeting epithelial receptors for enhanced oral vaccine delivery, J. Control. Release, 160 (2012) 431-439. [3] V. Melkebeek, K. Rasschaert, P. Bellot, K. Tilleman, H. Favoreel, D. Deforce, B.G. De Geest, B.M. Goddeeris, E. Cox, Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response, Mucosal Immunol., 5 (2012) 635-645. [4] K. Baert, B.G. de Geest, R. de Rycke, A.B. da Fonseca Antunes, H. de Greve, E. Cox, B. Devriendt, β-glucan microparticles targeted to epithelial APN as oral antigen delivery system, J. Control. Release, 220 (2015) 149-159. [5] B. Narasimhan, J.T. Goodman, J.E. Vela Ramirez, Rational design of targeted next-generation carriers for drug and vaccine delivery, Annu. Rev. Biomed. Eng., 18 (2016) 25-49. [6] W.W. Cheng, T.M. Allen, The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment, Expert Opin. Drug Deliv., 7 (2010) 461-478. [7] C.R. Robinson, R.T. Sauer, Optimizing the stability of single-chain proteins by linker length and composition mutagenesis, Proc. Natl. Acad. Sci. USA, 95 (1998) 5929-5934. [8] J.N. Duarte, J.J. Cragnolini, L.K. Swee, A.M. Bilate, J. Bader, J.R. Ingram, A. Rashidfarrokhi, T. Fang, A. Schiepers, L. Hanke, H.L. Ploegh, Generation of immunity against pathogens via single-domain antibody-antigen constructs, J. Immunol., 197 (2016) 4838-4847. [9] T. De Meyer, S. Muyldermans, A. Depicker, Nanobody-based products as research and diagnostic tools, Trends Biotechnol., 32 (2014) 263-270.

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Claims

1. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD), wherein said ISVD is a variable domain of a heavy chain antibody (VHH), wherein said ISVD specifically binds to CD13 and comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (a) CDR1 consists of the amino acid sequence represented by SEQ ID NO: 1, CDR2 consists of the amino acid sequence represented by SEQ ID NO: 3, and CDR3 consists of the amino acid sequence represented by SEQ ID NO: 4; or (b) CDR1 consists of the amino acid sequence represented by SEQ ID NO: 2, CDR2 consists of the amino acid sequence represented by SEQ ID NO: 3, and CDR3 consists of the amino acid sequence represented by SEQ ID NO: 5; The polypeptide.

2. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO:6 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

6.

3. The polypeptide is selected from the group consisting of:

3. The polypeptide of claim 1 or 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

4. The polypeptide of any one of claims 1 to 3, further comprising an Fc domain.

5. A construct comprising a polypeptide according to any one of claims 1 to 4 and a compound.

6. The construct described in claim 5, wherein the compound is a physiologically active compound or a diagnostic agent.

7. The construct described in claim 6, wherein the physiologically active compound is an antigen, a therapeutic agent or a toxin.

8. The construct described in claim 7, wherein the antigen is a protein, peptide, lipid, nucleic acid, glycolipid, glycoprotein, carbohydrate, oligosaccharide or polysaccharide.

9. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 4.

10. A host cell comprising the nucleic acid of claim 9.

11. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 4, a construct according to any one of claims 5 to 8, a nucleic acid according to claim 9, or a host cell according to claim 10, and a pharmaceutically acceptable carrier, excipient and / or diluent.

12. A polypeptide according to any one of claims 1 to 4, a construct according to any one of claims 5 to 8, a nucleic acid according to claim 9, a host cell according to claim 10 or a pharmaceutical composition according to claim 11 for use as a medicament.

13. A polypeptide according to any one of claims 1 to 4, a construct according to any one of claims 5 to 8, a nucleic acid according to claim 9, a host cell according to claim 10, or a pharmaceutical composition according to claim 11 for use in mucosal vaccination.

14. 12. A polypeptide according to any one of claims 1 to 4, a construct according to any one of claims 5 to 8, a nucleic acid according to claim 9, a host cell according to claim 10, or a pharmaceutical composition according to claim 11 for use in treating or preventing a disease of the intestine.

15. A polypeptide according to any one of claims 1 to 4, a construct according to any one of claims 5 to 8, a nucleic acid according to claim 9, a host cell according to claim 10, or a pharmaceutical composition according to claim 11 for use in treating or preventing infection or inflammation of the gastrointestinal tract or intestines. (a) introducing the nucleic acid of claim 9 into a Pichia pastoris expression system; and (b) purifying the expressed polypeptide; A method for producing the polypeptide according to any one of claims 1 to 4, comprising:

17. A method for determining a polypeptide or small molecule that binds to CD13, the method comprising competing the polypeptide or small molecule with a polypeptide comprising an array represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

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