Enteral delivery of immunoglobulin single variable domains

Abstract

The present invention relates to immunoglobulin single variable domains (ISVDs) for the treatment of diseases by the enteral, e.g. oral, delivery. In particular, the present invention provides ISVDs comprising sequences with a high percentage of sequence identity to SEQ ID NO: 1 for such enteral administration. Such sequences were identified as extraordinarily stable in the gastrointestinal tract, which prevents their degradation and thereby allows them to exert a strong therapeutic effect.

Description

[0001] Enteral Delivery of Immunoglobulin Single Variable Domains

[0002] DESCRIPTION

[0003] 1 Field of the present invention

[0004] The present invention relates to enteral delivery of drugs comprising polypeptides, in particular immunoglobulin single variable domains (ISVDs). Enteral, in particular oral, delivery of ISVDs according to the present invention is possible due to the extraordinary stability in the gastrointestinal tract.

[0005] 2 Background

[0006] Oral delivery of polypeptide drugs, in particular of antibodies, has long been a goal in the pharmaceutical industry due to the numerous advantages it offers compared to other modes of administration. Oral administration would improve the comfort of the patient greatly, by avoiding any invasive handling like syringes, and enabling self-administration, hence eliminating the need for a trained administrator. This will improve patient compliance and has the potential to reduce the associated health care costs. This increased patient convenience and compliance makes oral administration well suited for chronic treatment. Importantly, the oral route may also result in less systemic toxicity and higher efficacy potential, in particular for gastro-intestinal diseases. Moreover, oral drugs may yield in reduced manufacturing, transport and storage costs and hence overall lower treatment costs.

[0007] However, many therapeutic polypeptides, including conventional antibodies, are unstable in the intestinal tract, limiting the beneficial effects of oral administration. Various strategies have been employed to overcome this challenge, including formulation vehicles, enzyme inhibitors, absorption enhancers, and mucoadhesive polymers. Additionally, modifications to the therapeutic polypeptides themselves, have been explored with mixed results. The present invention focuses on the use of molecules comprising immunoglobulin single variable domains (ISVDs) for oral delivery. ISVDs offer unique advantages over conventional antibodies, including their smaller size and potential for increased stability. However, the challenge of maintaining stability in the gut environment while preserving binding affinity and potency has remained a significant hurdle also for ISVDs. To overcome those limitations, the inventors of the present invention have identified ISVDs which has an exceptionally high stability under the conditions of the Gl tract and are thus suitable for enteral, in particular oral, administration. This important discovery opens up new possibilities for the treatment of various diseases, particularly focusing on gastrointestinal tract disorders, through the convenient and patient-friendly oral route of administration.

[0008] 3 Summary of the present invention

[0009] The present invention focusses on a novel approach for the delivery of molecules comprising ISVDs, specifically ISVDs with high sequence identity to SEQ ID NO: 1. While ISVD sequences with high sequence identity to SEQ ID NO: 1 are known in the art, for example in WO2015173325, their stability and efficacy when administered enterally, e.g. orally, have not been previously recognized or demonstrated. The present invention reveals the unexpected stability of these ISVD molecules in the gut environment for the first time.

[0010] Enteral delivery of the ISVDs described in this invention may offer at least one or more of the following advantages:

[0011] • Improved patient comfort and compliance

[0012] • Reduced dosage requirements compared to traditional injectable therapies, in particular for diseases of the Gl tract

[0013] • Avoiding side effects linked to a systemic administration of a drug, in particular for diseases of the Gl tract

[0014] • Avoiding adverse reactions at the injection site Potential for reduced manufacturing, transport and storage costs and hence overall lower treatment costs

[0015] The present invention provides the following exemplary embodiments:

[0016] 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0017] 2. The molecule for use according to the previous embodiment, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0018] 3. The molecule for use according to any of the previous embodiments, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0019] 4. The molecule for use according to any of the previous embodiments, wherein the molecule consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0020] 5. The molecule for use according to any of the previous embodiments, wherein the ISVD binds to tumor necrosis factor (TNF).

[0021] 6. The molecule for use according to any of the previous embodiments, where the molecule is administered orally, sublingually, sublabial, buccal, rectally or via an ostomy system to the subject.

[0022] 7. The molecule for use according to any of the previous embodiments, where the molecule is administered orally to the subject.

[0023] 8. The molecule for use according to any of the previous embodiments, wherein the disease is a systemic disease or a disease of the gastrointestinal tract.

[0024] 9. The molecule for use according to any of the previous embodiments, wherein the disease is a disease of the gastrointestinal tract, optionally selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, and infectious diseases of the Gl tract.

[0025] 10. The molecule for use according to any of the previous embodiments 1-3 and 5-9, wherein the molecule comprises one or more additional ISVDs.

[0026] 11. The molecule for use according to the previous embodiment, wherein the ISVD which has at least 80% identity to SEQ ID NO: 1 and the one or more additional ISVDs are linked by one or more linkers, such as peptidic linkers.

[0027] 12. The molecule for use according to any of the two previous embodiments 10 and 11, wherein the one or more additional ISVDs bind to a target selected from IL-23, IL-22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A, DR5, OX40L, and ICOS.

[0028] 13. The molecule for use according to any of the previous embodiments 1-3 and 5-12, wherein the molecule comprises one or more other groups, residues, moieties, or binding units, optionally linked by one or more linkers, such as peptidic linkers, wherein said one or more other groups, residues, moieties or binding units provide the molecule with increased half-life, increased gut permeability, and / or increased bioavailability, compared to the corresponding molecule without said one or more other groups, residues, moieties or binding units.

[0029] 14. The molecule for use according to the previous embodiment, wherein said one or more othergroups, residues, moieties, or binding units that provide the molecule with increased half-life, increased gut permeability, and / or increased bioavailability are chosen from the group consisting of a. binding units that can bind to serum proteins, b. serum proteins or fragments thereof, c. polyethylene glycol residues, d. binding units that bind to FcRn, e. binding units that bind a receptor for active transport across the gut epithelium, f. a portion of an active transport domain for transport across the gut epithelium, such as a portion of Cholix protein, and g. fatty acids.

[0030] 15. A composition comprising a molecule as defined in any of the previous embodiments for use in treating a disease in a subject, wherein the composition is administered enterally to the subject, wherein the composition comprises the molecule and one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, optionally comprises a protease inhibitor, and optionally comprises one or more further pharmaceutically active compounds.

[0031] 4 Brief description of the drawings

[0032] Figure 1: Schematic representation of the minipig model used in example 1. The ISVDs (NB) are administered in the Gl tract either intraduodenally via a catheter or intracecally via a T- fistula.

[0033] Figure 2: ISVD concentrations in serum (Fig. 2A) and terminal ileum contents (Fig. 2B) after intraduodenal administration in the minipig model. 4 ISVDs (NB1, NB2, NB3 and NB4) were tested, the experiments were performed in 5 (NB1, NB2) or 4 (NB3, NB4) individual minipigs. Shown is the mean concentration in ng / mL, including standard deviation, for each ISVD and each timepoint. LLOQ (lower limit of quantification) indicates the lower detection limit of the employed bioanalytical assay. Values below the LLOQ of assay were assigned an arbitrary value of 0.1 ng / mL for the purpose to plotting the data in the graphs.

[0034] Figure 3: ISVD concentrations in feces after intraduodenal administration in the minipig model. 4 ISVDs (NB1, NB2, NB3 and NB4) were tested, the experiments were performed in

[0035] 5 (NB1, NB2) or 4 (NB3, NB4) individual minipigs. Fig. 3A shows the mean concentration in ng / mL, including standard deviation, for each ISVD and each timepoint. Fig. 3B shows the total feces recovery in ng. Each symbol represents one individual minipig, the horizontal line indicates the median value. LLOQ (lower limit of quantification) indicates the lower detection limit of the employed bioanalytical assay for NB1, NB2 and NB3. For NB4, the LLOQ was 13.4 ng / mL. Values below the LLOQ of assay were assigned an arbitrary value of 0.1 ng / mL (Fig. 3A) or 1 ng (Fig. 3B) for the purpose of plotting the data in the graphs.

[0036] Figure 4: ISVD concentrations in serum (Fig. 4A) and feces (Fig. 4B) after intracecal administration in the minipig model. 2 ISVDs (NB1 and NB2) were tested, the experiments were performed in 5 (individual minipigs for each of NB1 and NB2. Shown is the mean concentration in ng / mL, including standard deviation, for each ISVD and each timepoint. LLOQ (lower limit of quantification) indicates the lower detection limit of the employed bioanalytical assay. Values below the LLOQ of assay were assigned arbitrary values of 0.1 ng / mL or 1 ng for the purpose of plotting the data in the graphs.

[0037] Figure 5: ISVD serum concentrations (Fig. 5A) and recovery of ISVD from gut contents and excreted feces (Fig. 5B and C) after intraduodenal administration in the rat model. 2 ISVDs (NB1 and NB2) were tested, the experiments were performed in 3 individual rats for each time point and ISVD (i.e. in total 18 rats per ISVD). Fig. 5A depicts the mean ISVD serum concentration in ng / mL, including standard deviation, for each ISVD and each timepoint. LLOQ (lower limit of quantification) indicates the lower detection limit of the employed bioanalytical assay. Fig. 5B shows the total ISVD recovery from the Gl tract, as measured in collected luminal contents from small intestine, cecum, colon, and excreted feces. Fig. 5C indicates the recovered percentage of the administrated dose, calculated as the total recovery shown in Fig. 5B divided by the administered dose.

[0038] Figure 6: ISVD serum concentrations (Fig. 6A) and recovery (Fig. 6B and C) after intracecal administration in the rat model. 2 ISVDs (NB1 and NB2) were tested, the experiments were performed in 3 individual rats for each time point and ISVD (i.e. in total 18 rats per ISVD). Fig. 6A depicts the mean ISVD serum concentration in ng / mL, including standard deviation, for each ISVD and each timepoint. LLOQ (lower limit of quantification) indicates the lower detection limit of the employed bioanalytical assay. Fig. 6B shows the total ISVD recovery from the Gl tract, as measured collected luminal contents from cecum and colon and excreted feces. Fig. 6C indicates the recovered percentage of the administrated dose, calculated as the total recovery shown in Fig. 6C divided by the administered dose.

[0039] Figure 7: Graph depicting changes in the mean group body weight during the in vivo study (Day 0 to Day 13). Body weights are expressed as percentage of the initial value of the respective mice at day 0. Each study group is represented by a line (each data point is mean of 8 to 10 mice from the respective groups). Error bars indicate the standard error of the mean.

[0040] Figure 8: Graph Bars depicting levels of mlL6 in pg / ml at the end of the study on day 13 detected by ELISA in plasma samples of different study groups (each dot represents a mouse). Error bars indicate the standard error of the mean.

[0041] Figure 9: Graph Bars depicting the aggregated colon histopathology score (extent of inflammation + crypt damage + % involvement) for different study groups (each dot represents a mouse). Error bars indicate the standard error of the mean.

[0042] Figure 10: For the indicated intervals time points T (0, 2, 4 and 8 hours) post incubation in pig derived intestinal fluids (Jejunum, duodenum and ileum) at 37°C, NB1 and NB1-NB1 were analyzed in western blots using antibodies against ISVDs. Intensity of the respective bands in above blots is indicative of the stability of ISVDs in pig fluids.

[0043] 5 Detailed description of the present invention

[0044] The present invention provides molecules comprising ISVDs with high stability in the gastrointestinal tract for use in enteral delivery. The high stability of said ISVDs is linked to their amino acid sequence. Thus, the ISVD for use in the present invention have at least 80% identity to SEQ ID NO: 1.

[0045] To be suitable for enteral administration, a molecule comprising an ISVD has to meet the following criteria: (i) high intestinal stability while maintaining binding affinity and potency; (ii) high resistance to proteases found in the small and large intestine, including trypsin, chymotrypsin; (iii) high stability in the presence of proteases from gut commensal microflora and pathogenic bacteria. If the molecule is intended for the treatment of a systemic disease, it also has to have (iv) sufficient transport across the gut epithelium to ensure suitable bioactivity and biodistribution.

[0046] 5.1 Definitions

[0047] Unless otherwise stated below, all terms used in this application, including the specification and claims, have the meaning usually given to them in the respective scientific field.

[0048] As used in the specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents unless the context clearly dictates otherwise.

[0049] The term "immunoglobulin single variable domain" (ISVD), as used herein, interchangeably used with "single variable domain", defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab')2, scFv, di-scFv), wherein two immunoglobulin domains, in particulartwo variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0050] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0051] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain.

[0052] As such, the 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 VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the 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).

[0053] An immunoglobulin single variable domain (ISVD) can for example be a heavy-chain ISVD, such as a VHH, including a humanized VHH, a VH, including a camelized VH and a human VH. In one embodiment, it is a VHH, a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0054] For example, the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a NANOBODY® ISVD (as defined herein, and including but not limited to a VHH); other single variable domains, such as VNARs, or any suitable fragment of any one thereof.

[0055] In particular, the immunoglobulin single variable domain may be a NANOBODY® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof. [Note: NANOBODY ® and NANOBODIES® are registered trademarks of Ablynx N.V.]

[0056] "VHH domains", also known as VHHs, VHH antibody fragments, have originally been described as the antigen binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al. Nature 363: 446-448, 1993). The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHH's, reference is made to the review article by Muyl derma ns (Reviews in Molecular Biotechnology 74: 277-302, 2001).

[0057] The generation of immunoglobulin sequences, such as VHHs, has been described extensively in various publications, among which WO1994 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). In these methods, camelids are immunized with the target antigen in order to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind the target antigen.

[0058] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0059] Immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences can be used in the present technology. Also, fully human, humanized or chimeric sequences can be used. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al (see for example WO1994 / 04678, Davies and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996) can be used herein. The ISVDs can be fused forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350), as well as to for example WO1996 / 34103 and WO1999 / 23221). However, it should be noted that the ISVD comprised in the present technology is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In a specific but nonlimiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized camelid, mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized VH sequences), as well as ISVDs that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

[0060] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

[0061] A "humanized VHH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized" , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the prior art (e.g. W02008 / 020079). Again, it should be noted that such humanized VHHs can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0062] A "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a (camelid) heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description in the prior art (e.g. Davies and Riechman (1994 and 1996), supra). Such "camelizing" substitutions are inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so- called Camelidae hallmark residues, as defined herein (see for example WO1994 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0063] The structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions ("FRs"), which are referred to in the art and herein as "Framework region 1" ("FR1"); as "Framework region 2" ("FR2"); as "Framework region 3" ("FR3"); and as "Framework region 4" ("FR4"), respectively; which framework regions are interrupted by three complementary determining regions ("CDRs"), which are referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"); as "Complementarity Determining Region 2" ("CDR2"); and as "Complementarity Determining Region 3" ("CDR3"), respectively.

[0064] As further described in paragraph q) on pages 58 and 59 of W02008 / 020079, the amino acid residues of an ISVD can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, 1999 (J. Immunol. Methods 231: 25-38; see for example Figure 2 of this publication). It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0065] In the present application, unless indicated otherwise, CDR sequences were determined according to the AbM numbering as described in Kontermann and Dubel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter s, pp. 33- 51). According to this method, FR1 of an ISVD comprises the amino acid residues at positions 1-25, CDR1 of an ISVD comprises the amino acid residues at positions 26-35, FR2 of an ISVD comprises the amino acids at positions 36-49, CDR2 of an ISVD comprises the amino acid residues at positions 50-58, FR3 of an ISVD comprises the amino acid residues at positions 59-94, CDR3 of an ISVD comprises the amino acid residues at positions 95-102, and FR4 of an ISVD comprises the amino acid residues at positions 103-113.

[0066] Determination of CDR regions may also be done according to different methods. In the CDR determination according to Kabat, FR1 of an ISVD comprises the amino acid residues at positions 1-30, CDR1 of an ISVD comprises the amino acid residues at positions 31-35, FR2 of an ISVD comprises the amino acids at positions 36-49, CDR2 of an ISVD comprises the amino acid residues at positions 50-65, FR3 of an ISVD comprises the amino acid residues at positions 66-94, CDR3 of an ISVD comprises the amino acid residues at positions 95-102, and FR4 of an ISVD comprises the amino acid residues at positions 103-113.

[0067] The framework sequences are (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a VL-sequence) and / or from a heavy chain variable domain (e.g. a VH- sequence or VHH sequence). In one particular aspect, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).

[0068] In particular, the framework sequences present in the ISVD sequence used in the methods described herein may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a NANOBODY® ISVD, such as e.g. a VHH, including a humanized VHH, or camelized VH. Non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0069] Generally, NANOBODY® ISVDs (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein). Thus, generally, a NANOBODY® ISVD can be defined as an immunoglobulin sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0070] In particular, a NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure

[0071] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0072] More in particular, a NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure

[0073] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table 1 below. Table 1: Hallmark Residues in NANOBODY® ISVDs

[0074] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions effective in preventing or reducing binding of so- called "pre-existing antibodies" to the ISVD. ISVDs in which (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably

[0075] V have been described in WO2015 / 173325.

[0076] Digestive tract, as used herein is the digestive system which consists of the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum) and anus.

[0077] Gastrointestinal tract, or Gl tract, as used herein, includes the stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum) and anus.

[0078] Enteral Administration, as used herein, is the administration via the digestive tract or through any route that involves absorption by the digestive tract. This includes, but is not limited to: (i) Oral administration (through the mouth and swallowing); (ii) sublingual administration (under the tongue) (iii) sublabial administration (between the lip and gum); (vi) buccal administration (between the cheek and gum); (v) rectal administration (through the rectum); (vi) administration via an ostomy system (such as a bag for ostomy, for example gastrostomy or jejunostomy). In some embodiments, the enteral administration is administration via the Gl tract or through any route that involves absorption by the Gl tract. Enteral administration allows for the substance to come into contact with the digestive tract, either for local effect or for absorption into the systemic circulation. This route of administration is distinct from parenteral administration, which bypasses the digestive tract entirely.

[0079] Oral Administration, as used herein, is the introduction of a substance into the body through the mouth and swallowing, allowing it to enter the Gl tract.

[0080] Tumor necrosis factor, or TNF, as used herein, is a cell signaling protein (cytokine) involved in systemic inflammation. TNF plays a crucial role in the immune system's response to infection and tissue damage. It promotes inflammation, inhibits tumor growth, and can induce apoptosis. TNF is involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Anti-TNF antibodies have been successfully used as therapeutic agents in these conditions, demonstrating significant efficacy in reducing inflammation and improving patient outcomes. However, current anti-TNF therapies are typically administered via injection, highlighting the need of an orally administered anti-TNF antibody, such as an anti-TNF ISVD. TNF is a key target forthe ISVD in the molecules for administration according to the present invention.

[0081] A systemic disease, as used herein, is a disease that affects multiple organs or the entire body, as opposed to a localized disease affecting only one part of the body.

[0082] A disease of the gastrointestinal tract, as used herein, is any disease that affects the gastrointestinal tract. Examples of diseases of the gastrointestinal tract are inflammatory bowel disease (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), celiac disease, irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD), eosinophilic gastrointestinal disease peptic, ulcer disease, diverticulitis, gastrointestinal cancers (e.g., colorectal cancer, stomach cancer), short bowel syndrome, intestinal ischemia, gastroparesis, microscopic colitis, Whipple's disease, Hirschsprung's disease, and intestinal pseudo-obstruction.

[0083] Bioavailability, as used herein, is the proportion of a drug or other substance which enters the circulation when introduced into the body and so is able to have an active effect. Sufficient bioavailability is crucial for enterally administered drugs that are intended to have an effect against a systemic disease.

[0084] Increased Bioavailability, as used herein, refers to an enhancement in the fraction of the administered dose that reaches the systemic circulation in an active form after enteral administration. This can be achieved through various means, including protection from degradation in the Gl tract, increased gut permeability, or modifications that improve absorption.

[0085] Gut permeability, as used herein, refers to the ability of a molecule to pass through the intestinal epithelium ("gut wall") and to be absorbed into the bloodstream or lymphatic system. Increased gut permeability, as used herein, refers to an enhanced ability of a molecule to pass through the intestinal epithelium and enter the bloodstream after enteral administration. This can be achieved through various strategies, such as the use of permeation enhancers or modification of the molecule structure.

[0086] Increased half-life, as used herein, refers to an extension of the time it takes for the concentration of a molecule to reduce by half after enteral administration. An increase halflife can be achieved through various modifications of the molecule or its formulation, resulting in prolonged therapeutic effect and potentially reduced dosing frequency. Exemplary molecules which can increase the half-life of a molecule are fusion proteins with an albumin-binding ISVD, fusion proteins with an FcRn-binding ISVD and / or fusion proteins with an Fc region of an antibody.

[0087] A subject, as used herein, can be any animal, and more specifically a mammal. Among mammals, there are humans and non-human mammals. Non-human animals may be for example companion animals (e.g. dogs, cats), livestock (e.g. bovine, equine, ovine, caprine, or porcine animals), or animals used generally for research purposes and / or for producing antibodies (e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llama or alpaca). In one embodiment, the subject is a human subject.

[0088] 5.2 ISVD for enteral administration.

[0089] The present invention provides molecules comprising an ISVD for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0090] SEQ ID NO: 1 is the following sequence:

[0091] DVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKG RFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS.

[0092] The ISVD has been found to display an exceptionally high stability in the gastrointestinal tract (see examples below). In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the ISVD has at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the ISVD has at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the ISVD has at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the ISVD has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the ISVD is SEQ ID NO: 1 (i.e., consisting of SEQ ID NO: 1). or SEQ ID NO: 2

[0093] (i.e., consisting of SEQ ID NO: 2). In some embodiments, the ISVD is SEQ ID NO: 1 (i.e., consisting of SEQ ID NO: 1). In some embodiments, the ISVD is SEQ ID NO: 2 (i.e., consisting of SEQ ID NO: 2). SEQ ID NO: 2 is identical to SEQ ID NO: 1 except of an amino acid exchange on position 1, where SEQ ID NO: 1 has D, while SEQ ID NO: 2 has E. Thus, SEQ ID NO: 2 has more than 99% sequence identity to SEQ ID NO: 1.

[0094] The CDRs of SEQ ID NO: 1 are given in Table 2 below, according to the most common definitions.

[0095] Table 2: CDRs of SEQ ID NO: 1 according to IMGT, Kabat and AbM definition. The definitions are according to the commonly known CDR annotations rules as known in 2024, as found e.g. on http: / / bioinf.org.uk / abs / info.html.

[0096] The ISVD of SEQ ID NO: 2 has identical CDRs as the ISVD of SEQ ID NO: 1.

[0097] In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence GFTFSDYW, a CDR2 that is the amino acid sequence INTNGLIT, and a CDR3 that is the amino acid sequence ARSPSGFN, wherein the CDRs are defined according to the IMGT definition. In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:3, a CDR2 that is the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:4, and a CDR3 that is the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:5, wherein the CDRs are defined according to the IMGT definition.

[0098] In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence DYWMY, a CDR2 that is the amino acid sequence EINTNGLITKYPDSVKG, and a CDR3 that is the amino acid sequence SPSGFN, wherein the CDRs are defined according to the Kabat definition. In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:6, a CDR2 that is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:7, and a CDR3 that is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:8, wherein the CDRs are defined according to the Kabat definition.

[0099] In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence GFTFSDYWMY, a CDR2 that is the amino acid sequence EINTNGLITK, and a CDR3 that is the amino acid sequence SPSGFN, wherein the CDRs are defined according to the AbM definition. In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0100] The ISVD of SEQ ID NO: 1 binds to TNF. The ISVD of SEQ ID NO: 2 also binds also to TNF.

[0101] In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and binds to TNF. In some embodiments, the ISVD has at least 80% sequence identity to SEQ ID NO: 1, comprises a CDR1 that is the amino acid sequence GFTFSDYW, a CDR2 that is the amino acid sequence INTNGLIT, and a CDR3 that is the amino acid sequence ARSPSGFN, wherein the CDRs are defined according to the IMGT definition, and binds to TNF.

[0102] In one embodiment, the ISVD has at least 80% sequence identity to SEQ ID NO: 1 and further comprises a C-terminal extension, such as a C-terminal alanine (A) or glycine (G) extension.

[0103] In one embodiment, the ISVD has at least 80% sequence identity to SEQ ID NO: 24. SEQ ID NO: 24 is SEQ ID NO: 1 with a C-terminal alanine extension. In one embodiment, the ISVD has at least 90% sequence identity to SEQ ID NO: 24. In one embodiment, the ISVD has at least 95% sequence identity to SEQ ID NO: 24. In one embodiment, the ISVD has at least 99% sequence identity to SEQ ID NO: 24. In one embodiment, the ISVD is SEQ ID NO: 24. In one embodiment, the ISVD is SEQ ID NO: 24, wherein the D at position 1 is substituted by an E.

[0104] 5.3 Molecules of the present invention

[0105] The molecules for use according to the present invention comprise an ISVD as defined above. The molecules may consist only of this ISVD or comprise further elements, such as additional ISVDs, other groups, residues, moieties, or binding units. Those other ISVDs groups, residues, moieties, or binding units can either bind to a target to exert a pharmacodynamic effect (in addition to the pharmacodynamic effect of the ISVD) or improve the properties of the whole molecule, such as increased half-life, increased gut permeability and / or increased bioavailability. In some embodiments, the molecule of the present invention is a polypeptide.

[0106] 5. 3.1 Additional targets for a mic effect

[0107] In some embodiments, the molecule comprises one or more additional ISVDs, or other binding units, which bind to targets to exert a pharmacodynamic effect. Said ISVDs, or other binding units, can bind to any target which plays a role in the disease to be treated and whose inhibition or activation can contribute to the treatment of said disease. In some embodiments, the targets or the one or more ISVD, or other binding units, are selected from interleukin-23 (IL-23), interleukin-22 (IL-22), TNF, alpha4 betal integrin, alpha4 beta? integrin, interleukin-15 (IL-15), interleukin-21 (IL-21), interleukin-6 (IL-6), interleukin-1 (I L- 1), interferon-gamma (IFN-g), transforming growth factor-beta (TGF-b), vascular endothelial growth factor (VEGF), Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A,DR5, OX40L, and ICOS. In some embodiments, the molecule comprises at least one additional ISVD and the target of said additional ISVD is selected from IL-23, IL-22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TLlA, DR5, OX40L, and ICOS. In some embodiments, the molecule comprises at least one additional ISVD and the target of said additional ISVD is the same target as the target of the ISVD which has at least 80% identity to SEQ ID NO: 1. In some embodiments, the molecule comprises at least one additional ISVD and the target of said additional ISVD is the same target as the target of the ISVD which has at least 80% identity to SEQ ID NO: 1, wherein the two ISVDs bind to the target at different epitopes.

[0108] In some embodiments, the molecule comprises at least two additional ISVD and the targets of said additional two ISVD are selected from IL-23, IL-22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A,DR5, OX40L, and ICOS. In some embodiments, the molecule comprises at least two additional ISVD and the targets of said additional two ISVD are selected from IL-23, IL-22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A,DR5, OX40L, and ICOS, wherein the target of said additional two ISVDs is the same. In some embodiments, the molecule comprises at least two additional ISVD and the targets of said additional two ISVD are selected from IL-23, IL- 22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A,DR5, OX40L, and ICOS, wherein the target of said additional two ISVDs is the same, wherein the two additional ISVDs bind to the target at different epitopes.

[0109] In some embodiments, the molecule comprises one or more binding unit, other than an ISVD, which bind to targets to exert a pharmacodynamic effect. Exemplary binding units are antibodies, antibody fragments, Ffab'^s, Fabs, scFvs, cytokines, hormones, and other pharmaceutically active peptides.

[0110] 5.3.2 Groups, residues, moieties, and binding units improved molecular properties

[0111] The molecule of the present invention may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers, such as peptidic linkers, to the ISVD which has at least 80% identity to SEQ ID NO: 1 and / or additional ISVDs as described above. These other groups, residues, moieties or binding units are designed to provide the molecule with one or more of the following enhanced properties, compared to the corresponding molecules without said other groups, residues, moieties or binding units: increased half-life, increased gut permeability, and / or increased bioavailability.

[0112] 5.3.2.1 Increased half-life

[0113] Half-life extension means, for example, that the molecule has an increased half-life in a mammal, such as a human subject, after enteral administration.

[0114] The type of groups, residues, moieties, or binding units for half-life extension is not generally restricted and may be chosen from the group consisting of: a) binding units that can bind to serum proteins, b) serum proteins or fragments thereof, c) polyethylene glycol residues, d) binding units that bind to FcRn.

[0115] In particular, said one or more other groups, residues, moieties, or binding units that provide the molecule with increased half-life can be chosen from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG.

[0116] In one embodiment, said one or more other binding units that provide the molecule with increased half-life is a binding unit that can bind to human serum albumin. In one embodiment, the binding unit is an ISVD.

[0117] For example, W02004 / 041865 describes NANOBODY® molecules binding to serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other Nanobodies binding to a desired target) in order to increase the half-life of said protein. The international application WO2006 / 122787 describes a number of ISVDs against (human) serum albumin. These ISVDs include the ISVD called Alb-1 (SEQ ID NO: 52 in WO2006 / 122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO2006 / 122787). Moreover, W02012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0118] In some embodiments, the molecule comprises a serum albumin binding ISVD selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23. In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23 or its variants, as shown in pages 7-9 of W02012 / 175400 and the albumin binders described in WO2012 / 175741, WO2015 / 173325, W02017 / 080850, WO2017 / 085172, WO2018 / 104444,

[0119] WO2018 / 134235, WO2018 / 134234.

[0120] In some embodiments, the molecule comprises a serum albumin binding ISVD which comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and a CDR3 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 14, wherein the CDRs are defined according to the AbM definition.

[0121] In some embodiments, the molecule comprises a serum albumin binding ISVD which comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 12; a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and a CDR3 that is the amino acid sequence of SEQ ID NO: 14, wherein the CDRs are defined according to the AbM definition. In some embodiments, the molecule comprises a serum albumin binding ISVD which has a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 14, wherein the CDRs are as defined in the previous embodiments. In some embodiments, the molecule comprises a serum albumin binding ISVD comprising or consisting of the full amino acid sequence of construct ALB23002 (SEQ ID NO: 15).

[0122] In some embodiments, the molecule comprises serum proteins or fragments thereof. Most common is the fusion of the molecule to human serum albumin (HSA) or fragments thereof, or to immunoglobulins or fragments thereof. Albumin, with its 19-day half-life in humans, is an attractive fusion partner for extending the circulatory time of therapeutics. Fc regions of IgG are also commonly used, as they can bind to the neonatal Fc receptor (FcRn), which protects them from lysosomal degradation. These fusion proteins combine the therapeutic activity of the pharmacodynamically active part of the molecule (e.g. an ISVD) with the pharmacokinetic advantages of the serum protein.

[0123] In some embodiments, the molecule comprises polyethylene glycol (PEG) residues. Those PEG residues are covalently attached to the molecule (PEGylation). PEG is a hydrophilic, inert polymer that forms a hydrated shell around the molecule, increasing its hydrodynamic radius and thus reducing renal clearance. PEGylation also shields the molecule from proteolytic enzymes and reduces immunogenicity. The size and branching of PEG can be tailored to achieve desired pharmacokinetic properties.

[0124] In some embodiments, the molecule comprises binding units, such as ISVDs, that bind to the neonatal Fc receptor (FcRn). FcRn plays a crucial role in protecting IgG and albumin from degradation by binding them in acidic endosomes and recycling them back to the circulation. By mimicking this interaction, binding units that target FcRn can extend the half-life of attached therapeutic molecules. This approach allows for half-life extension without the need for a full Fc region.

[0125] 5.3.2.2 Increased Gut Permeability

[0126] The molecule may comprise one or more other groups, residues, moieties, or binding units that provide the molecule with increased gut permeability, compared to the corresponding molecule without said one or more other groups, residues, moieties, or binding units. Increased gut permeability means, for example, that the molecule has an enhanced ability to cross the intestinal epithelial barrier in a mammal, such as a human subject, after enteral, such as oral, administration.

[0127] There are various types of groups, residues, moieties or binding units for increasing gut permeability. Exemplary groups are listed below: a) binding units that bind a receptor for active transport across the gut epithelium b) a portion of an active transport domain for transport across the gut epithelium, such as a portion of Cholix protein or homolog proteins from mono-ADP-ribosyltransferase (mART) toxin family c) cell-penetrating peptides d) mucoadhesive polymers e) tight junction modulators f) fatty acid conjugates

[0128] In some embodiments, the molecule comprises binding units, such as ISVDs, that bind a receptor for active transport across the gut epithelium. Examples of such receptors include the vitamin B12 receptor and sodium-glucose linked transporters (SGLTs). By utilizing these natural transport pathways, the attached therapeutic molecule can be actively transported across the epithelial barrier. This approach can be highly specific and efficient. Examples of binding units are antibodies and antibody fragments, Fab fragments, scFv, single domain antibodies, ISVD, DARPins, Vitamin B12, folic acid, biotin and thiamine.

[0129] In some embodiments, the molecule comprises a portion of an active transport domain for transport across the gut epithelium, such as a portion of Cholix protein or homolog proteins from mono-ADP-ribosyltransferase (mART) toxin family. Such active transport domains are fragments of proteins that naturally undergo transcytosis across the intestinal epithelium. The Cholix protein, for example, is an exotoxin from Vibrio cholerae that can cross the intestinal barrier. By identifying and utilizing the specific domains responsible for this transport, it is possible to create chimeric molecules that combine the transport capabilities of Cholix with the therapeutic function of the pharmacodynamically active part of the molecule.

[0130] In some embodiments, the molecule comprises cell-penetrating peptides (CPPs). CPPs are short amino acid sequences that can facilitate the transport of various molecular cargoes across cellular membranes. In the context of gut permeability, CPPs can enhance the transcellular transport of therapeutic molecules across intestinal epithelial cells. Common examples include TAT peptide (derived from HIV), penetratin, and polyarginine sequences. CPPs can work through various mechanisms, including direct membrane translocation and endocytosis.

[0131] In some embodiments, the molecule comprises mucoadhesive polymers. Mucoadhesive polymers are materials that adhere to the mucus layer lining the intestinal epithelium, increasing the residence time of the therapeutic molecule in the gut. This prolonged contact time can enhance absorption by allowing more time for the drug to permeate the epithelial barrier. Common examples include chitosan, carbomers, and thiolated polymers. These polymers can also modulate tight junctions, potentially enhancing paracellular transport. Mucoadhesive polymers can be used as coatings, conjugates, or as part of more complex delivery systems like nanoparticles.

[0132] In some embodiments, the molecule comprises tight junction modulators. Tight junction modulators are compounds that can reversibly open the tight junctions between intestinal epithelial cells, enhancing paracellulartransport of therapeutic molecules. One well-known example is zonula occludens toxin (ZOT) and fragments thereof, which interact with the ZOT receptor to induce tight junction disassembly. Other modulators include certain medium-chain fatty acids and chitosan derivatives.

[0133] In some embodiments, the molecule comprises fatty acids which are covalently attached to the molecule. Fatty acids can enhance transcellular absorption through increased lipophilicity and potential interactions with the intestinal epithelium. Common fatty acids used include caprylic acid (C8) and capric acid (CIO). The fatty acid conjugation can also provide some protection against enzymatic degradation in the gastrointestinal tract.

[0134] 5.3.2.3 Increased Bioavailability

[0135] The molecule may further comprise one or more other groups, residues, moieties, or binding units that provide the molecule with increased bioavailability, compared to the corresponding molecule without said one or more other groups, residues, moieties, or binding units. Increased bioavailability means that the molecule has an enhanced systemic exposure in a mammal, such as a human subject, after administration. Increased bioavailability can be caused by increased gut permeability, increased stability and increased half-life, thus the modifications described above for those aspects are also suitable to increase bioavailability.

[0136] 5.3.3 Linkers

[0137] The molecule can further comprise linkers, such as peptidic linkers, which links elements of the molecules, such as the ISVD which has at least 80% identity to SEQ ID NO: 1, one or more additional ISVDs, and / or other groups, residues, moieties, and binding units.

[0138] In some embodiments, the linkers are not cleavable by proteases which are present in the digestive tract. Such stable linkers are designed to resist proteolytic cleavage, maintaining the integrity of the multi-domain construct in the digestive tract. These stable linkers often consist of glycine and serine residues, which provide flexibility while minimizing susceptibility to proteases. Common examples of stable linkers include:

[0139] (GGGGS)n (SEQ ID NO: 16), where n is typically 1 to 5

[0140] (GGGGS)nGG (SEQ ID NO: 17), where n is typically 1 to 5

[0141] GSGSGS (SEQ ID NO: 18).

[0142] In some embodiments, the stable linkers essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 79) motif (for example, have the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more). Some often-used examples of such GS linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 80), 15GS linkers (n=3; SEQ ID NO: 81) and 35GS linkers (n=7; SEQ ID NO: 78). Reference is for example made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325-330). In one embodiment, 9GS linkers to link the components of the polypeptide to each other, are used. In another embodiment, 35GS linkers to link the components of the polypeptide to each other, are used. An example of ISVD formats with a 35GS linker is the molecule NB1- NB1 as given in SEQ ID NO: 76.

[0143] These glycine-serine rich linkers are generally resistant to trypsin, chymotrypsin, and other common gastrointestinal proteases due to the absence of their preferred cleavage sites.

[0144] In some embodiments, the linkers are cleavable by proteases which are present in the digestive tract. Cleavable linkers are designed to be susceptible to specific proteases present in the gastrointestinal tract. Those proteases include for example trypsin, trypsinlike proteases, chymotrypsin, chymotrypsin-like proteases, enteropeptidase, carboxypeptidase A, and elastase. Protease-cleavable linkers can be used to release parts of the molecules at desired locations orto modulate the pharmacokinetics of the molecule. Common examples of cleavable linkers include:

[0145] Trypsin-cleavable linkers with lysine or arginine residues followed by non-proline amino acids, for example:

[0146] (GGGGS)mK(GGGGS)n, where m and n are typically 1 to 5 (SEQ ID NO: 19); (GGGGS)mR(GGGGS)n, where m and n are typically 1 to 5 (SEQ ID NO: 20).

[0147] Chymotrypsin-cleavable linkers with an aromatic amino acids like phenylalanine, tyrosine, or tryptophan followed by non-proline residues, for example:

[0148] (GGGGS)mF(GGGGS)n, where m and n are typically 1 to 5 (SEQ ID NO: 21);

[0149] (GGGGS)mY(GGGGS)n, where m and n are typically 1 to 5 (SEQ ID NO: 22).

[0150] Enteropeptidase-cleavable linkers comprising the amino acids sequence DDDDK, followed by non-proline residues, for example:

[0151] (GGGGS)mDDDDK(GGGGS)n, where m and n are typically 1 to 5 (SEQ ID NO: 23).

[0152] The choice between stable and cleavable linkers depends on the desired pharmacokinetic profile and mechanism of action of the therapeutic molecule. Stable linkers are preferred when maintaining the intact structure throughout the gastrointestinal tract is crucial. Cleavable linkers, on the other hand, can be used to achieve targeted release, modulate absorption, or activate prodrugs.

[0153] 5.4 Enteral administration

[0154] The ISVD in the molecules used in the present invention are uniquely suited for enteral administration. This route of administration offers several advantages:

[0155] • Improved patient acceptance and compliance due to ease of use

[0156] • Improved potential for home-based treatment due to ease of use

[0157] • Reduced dosage requirements compared to traditional injectable therapies, in particular for diseases of the Gl tract

[0158] • Potential for localized treatment of gastrointestinal diseases, thus avoiding side effects linked to a systemic administration of a drug

[0159] • Avoiding adverse reactions at the injection site and other injection-related complications Potential for reduced production costs and improved yield during recombinant production

[0160] There are several different options for enteral administration, including oral administration, sublingual administration, sublabial administration, buccal administration, rectal administration, administration via an ostomy system, such as a bag for ostomy, intestinal administration, gastric administration, and duodenal administration.

[0161] In some embodiments, the enteral is oral administration. Oral administration is the most common and widely used route of enteral drug delivery. It involves ingesting the drug through the mouth, typically in the form of tablets, capsules, liquids, suspensions, devices for oral delivery, or other oral dosage forms. Oral delivery can be implemented using a variety of formulation techniques known in the art, such as controlled-release coatings, enteric coatings, or mucoadhesive formulations. The convenience and non-invasive nature of oral administration make it a preferred choice for many therapeutic agents, including the molecules described in the present invention.

[0162] In some embodiments, the enteral is sublingual administration. Sublingual administration involves placing the drug under the tongue, where it is absorbed through the mucous membranes into the bloodstream. This method is particularly useful for drugs that are susceptible to first-pass metabolism or degradation in the gastrointestinal tract. Sublingual formulations, such as tablets, films, or sprays, are well-established in the pharmaceutical industry and can be prepared using known excipients and manufacturing processes. The rapid absorption and onset of action associated with sublingual administration make it an attractive option for certain therapeutic applications.

[0163] In some embodiments, the enteral is sublabial administration. Sublabial administration refers to the placement of a drug between the lip and gum, allowing for absorption through the oral mucosa. This method shares similarities with sublingual and buccal administration and can be implemented using established formulation techniques such as mucoadhesive tablets or films. Sublabial administration is particularly useful for drugs that require prolonged contact with the oral mucosa for optimal absorption. In some embodiments, the enteral is buccal administration. Buccal administration involves placing the drug between the cheek and gum, where it is absorbed through the buccal mucosa. This route offers advantages such as avoiding first-pass metabolism and providing sustained drug release. Buccal formulations, including tablets, films, and patches, are well- established in the pharmaceutical industry and can be prepared using known mucoadhesive polymers and manufacturing techniques. The ability to provide controlled release and improved bioavailability can make buccal administration an attractive option for certain drugs.

[0164] In some embodiments, the enteral is rectal administration. Rectal administration involves delivering the drug through the rectum, typically using suppositories, enemas, or rectal gels. This route is particularly useful fordrugs that are poorly absorbed orally or for patients who cannot take medications by mouth. Rectal formulations are well-established and can be prepared using known bases and manufacturing processes. The rich blood supply in the rectal area and the ability to avoid first-pass metabolism make rectal administration an effective alternative for certain therapeutic agents.

[0165] In some embodiments, the enteral is administration via an ostomy system, such as a bag for ostomy. Administration via an ostomy system involves delivering the drug through a surgically created opening in the body, such as a gastrostomy or jejunostomy. The administration can occur by administering the molecule to the ostomy bag, ostomy stoma, or ostomy pouch. This method is particularly useful for patients who cannot take medications orally due to various medical conditions. Ostomy administration can be implemented using established techniques and devices, such as specialized feeding tubes and formulations designed for enteral nutrition. The direct access to specific parts of the gastrointestinal tract offers advantages for drug delivery in certain patient populations.

[0166] In some embodiments, the enteral is intestinal administration. Intestinal administration refers to the delivery of drugs directly to the small or large intestine, without being administered orally. This can be achieved through invasive methods like intestinal tubes or endoscopes. Intestinal administration is well-established in the pharmaceutical industry, with numerous formulation strategies available to target specific regions of the intestine. Intestinal administration can also be achieved via microbial delivery, i.e. the administration of microbes, such as bacteria, which produce and secrete the drug directly in the intestine. Intestinal administration is particularly useful for drugs that require site-specific delivery or for treating local intestinal conditions.

[0167] In some embodiments, the enteral is gastric administration. Gastric administration involves delivering the drug directly to the stomach, without being administered orally. This can be achieved through invasive methods like gastric tubes or endoscopes. Gastric administration is a well-known technique in pharmaceutics, with various formulation strategies available to enhance drug stability and absorption in the acidic environment of the stomach. This method is particularly useful for drugs that are best absorbed in the stomach or for treating gastric conditions.

[0168] In some embodiments, the enteral is duodenal administration. Duodenal administration involves delivering the drug directly to the duodenum, the first part of the small intestine, without being administered orally. This can be achieved through invasive methods like duodenal tubes or endoscopes. Duodenal administration is an established technique in pharmaceutical research and development, with various formulation strategies available to target this specific region of the gastrointestinal tract. Duodenal administration can also be achieved via microbial delivery, i.e. the administration of microbes, such as bacteria, which produce and secrete the drug directly in the duodenum. Duodenal administration is particularly useful for drugs that require a neutral pH for optimal absorption or for treating conditions specific to the duodenum.

[0169] 5.5 Disease treatment

[0170] The molecules for use according to the present invention can be used to treat a variety of diseases. In particular, the molecules of use according to the present invention can be used to treat a systemic disease or a disease of the gastrointestinal tract. The therapeutic effect can be mediated by binding to TNF, by binding to another target or by binding to TNF and another target. Systemic diseases

[0171] If the molecule for use according to the present invention enters the circulation after being administered enterally, e.g. by crossing the gut epithelium after oral administration, it can be used to treat a systemic disease. Exemplary systemic diseases which can be treated by enteral administration of molecules according to the present invention are rheumatoid arthritis (RA), psoriasis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), Type 1 diabetes, ankylosing spondylitis, Parkinson's disease, and Alzheimer's disease. The therapeutic effect for treatment of the systemic disease can be mediated by binding to TNF, by binding to another target or by binding to TNF and another target.

[0172] In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating rheumatoid arthritis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating rheumatoid arthritis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0173] Diseases of the gastrointestinal tract

[0174] Disease of the gastrointestinal tract can be treated in a very targeted manner by enteral administration of molecules according to the present invention, as the molecule can directly act in the Gl tract, i.e. its entry site in the body. Exemplary diseases of the Gl are inflammatory bowel disease (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), celiac disease, gluten intolerance, irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD), eosinophilic gastrointestinal disease, peptic ulcer disease, diverticulitis, gastrointestinal cancers (e.g., colorectal cancer, stomach cancer), short bowel syndrome, intestinal ischemia, gastroparesis, microscopic colitis, Whipple's disease, Hirschsprung's disease, and intestinal pseudo-obstruction, and infectious diseases of the Gl tract. The therapeutic effect for treatment of the systemic disease can be mediated by binding to TNF, by binding to another target or by binding to TNF and another target.

[0175] In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating inflammatory bowel disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. Inflammatory Bowel Disease (IBD) is an umbrella term primarily encompassing Crohn's disease and ulcerative colitis. IBD around 5-7 million people worldwide (Alatab et al., 2020; Wang et al., 2023).

[0176] It's characterized by chronic inflammation of the gastrointestinal tract. The exact cause is unknown, but it's believed to result from an abnormal immune response to the gut flora in genetically susceptible individuals. Symptoms can include abdominal pain, diarrhea, rectal bleeding, weight loss, and fatigue. Treatment options for IBD include anti-inflammatory drugs, immunosuppressants, biologies (like anti-TNF antibodies), and in severe cases, surgery. The goal of treatment is to induce and maintain remission, heal the intestinal mucosa, and improve quality of life.

[0177] In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. Crohn's disease (CD) is a type of IBD that can affect any part of the gastrointestinal tract from mouth to anus, but most commonly involves the end of the small intestine and the beginning of the colon. Crohn's disease causes patchy, discontinuous areas of inflammation that can affect all layers of the intestinal wall (transmural inflammation). Crohn's disease can be categorized based on the location of inflammation: ileocolitis, ileitis, gastroduodenal Crohn's disease, jejunoileitis, and Crohn's colitis. Symptoms include persistent diarrhea, abdominal pain, rectal bleeding, weight loss, and fatigue. Complications can include strictures, fistulas, and abscesses. Treatment options include corticosteroids for acute flares, immunomodulators, biologies (like anti-TNF antibodies), and surgery in cases of complications or treatment-resistant disease. The disease is characterized by periods of active illness followed by periods of remission.

[0178] In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. Ulcerative colitis is a type of IBD that affects the colon and rectum, causing inflammation and ulcers in the digestive tract. UC causes continuous inflammation that typically starts in the rectum and extends proximally, affecting only the innermost lining of the colon (mucosa and submucosa). Ulcerative colitis can be classified based on its extent: ulcerative proctitis (limited to the rectum), left-sided colitis, and extensive colitis or pancolitis. The main symptoms include rectal bleeding, diarrhea, abdominal pain, and urgency to defecate. Severity can range from mild to severe, with some patients experiencing life-threatening complications. Treatment options are similar to those for Crohn's disease and corticosteroids, immunomodulators, and biologies. In severe cases or when medical management fails, surgical removal of the colon (colectomy) may be necessary and can be curative for ulcerative colitis.

[0179] In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. Celiac disease is an autoimmune disorder triggered by the ingestion of gluten, a protein found in wheat, barley, and rye. It affects about 1% of the population worldwide, though many cases remain undiagnosed. When people with celiac disease eat gluten, it triggers an immune response that damages the small intestine's lining. This can lead to malabsorption of nutrients. Symptoms can vary widely but often include diarrhea, abdominal pain, bloating, fatigue, and weight loss. In children, it can also lead to growth problems. The only effective treatment for celiac disease is a strict, lifelong gluten-free diet. This allows the small intestine to heal and symptoms to resolve. If the small intestine is severely damaged, steroids may be administered to control inflammation while the intestine heals. In some embodiments, the present invention provides a molecule comprising an ISVD for use in treating an infectious disease of the Gl tract in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. Infectious diseases of the Gl tract are diseases which are caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi that affect the Gl tract. These infections can lead to various symptoms, including diarrhea, vomiting, abdominal pain, and fever. Examples of infectious diseases of the Gl tract are bacterial gastroenteritis, salmonellosis, campylobacteriosis, Helicobacter pylori infection, viral gastroenteritis, norovirus infection, rotavirus infection, and giardiasis.

[0180] 5.6 Formulations and compositions

[0181] The molecules for use according to the present invention can be formulated with one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants which are suitable for enteral administration.

[0182] Suitable formulations for enteral administration, particularly for oral delivery, are known in the field. Formulation components can be strategically selected to enhance drug solubility, stability, gut permeability, and ultimately, bioavailability. Common oral dosage forms include tablets, capsules, solutions, suspensions, and emulsions, each offering unique advantages for specific drug properties and therapeutic goals. Tablets and capsules, being solid dosage forms, often incorporate disintegrants to facilitate rapid dissolution in the gastrointestinal tract. Binders and fillers are used to improve the physical properties and manufacturability of solid dosage forms, while lubricants reduce friction during the manufacturing process. For poorly water-soluble drugs, solubilizers such as surfactants, cosolvents, or complexing agents (e.g., cyclodextrins) can significantly improve dissolution rates. pH-modifying agents can create a microenvironment that favors drug solubility and absorption, particularly for ionizable compounds. Controlled-release formulations, utilizing polymeric matrices or coatings, can modulate drug release profiles, improving therapeutic efficacy and reducing dosing frequency. Enteric coatings protect acid-labile drugs from gastric degradation and can target drug release to specific regions of the intestine. Mucoadhesive polymers can prolong gastrointestinal residence time, potentially enhancing absorption of drugs with narrow absorption windows. For macromolecular drugs like peptides and proteins, enzyme inhibitors can be incorporated to reduce pre-systemic metabolism. Permeation enhancers, such as medium-chain fatty acids or chelating agents, can improve the absorption of poorly permeable drugs. Nanoformulations, including lipid- based nanocarriers and polymeric nanoparticles, offer promising approaches for enhancing the oral bioavailability of challenging molecules.

[0183] Protease inhibitors play a crucial role in oral formulations of peptide and protein drugs, protecting these sensitive molecules from enzymatic degradation in the gastrointestinal tract and potentially enhancing their oral bioavailability. Common protease inhibitors used in oral formulations include aprotinin, soybean trypsin inhibitor, and synthetic inhibitors like nafamostat mesylate. Stomach-resistant enteric coatings are polymeric film coatings applied to solid dosage forms to prevent dissolution in the acidic environment of the stomach. These coatings, typically composed of pH-sensitive polymers such as cellulose acetate phthalate or methacrylic acid copolymers, remain intact at low pH but dissolve rapidly in the higher pH environment of the small intestine. This approach not only protects acid-labile drugs from degradation but also allows for targeted delivery to specific regions of the gastrointestinal tract, potentially improving therapeutic efficacy and reducing side effects. The selection of an appropriate enteric coating depends on factors such as the desired site of drug release, the drug's stability profile, and the intended dosing regimen.

[0184] In some embodiments, the present invention provides a composition comprising a molecule comprising an ISVD as defined above, wherein the composition is administered enterally to the subject, wherein the composition comprises the molecule and one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, and optionally comprises one or more further pharmaceutically active compounds. In some embodiments, the composition comprises a protease inhibitor. In some embodiments, the composition comprises an enteric coating. 5.7 Embodiments of the invention

[0185] In the following exemplary embodiments of the present invention are given for illustrative purposes.

[0186] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0187] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0188] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0189] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0190] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0191] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0192] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0193] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0194] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0195] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0196] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0197] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0198] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0199] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0200] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0201] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0202] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0203] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0204] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0205] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0206] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0207] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0208] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0209] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0210] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0211] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0212] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0213] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0214] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0215] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0216] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0217] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0218] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0219] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0220] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0221] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0222] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0223] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0224] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0225] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0226] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0227] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0228] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

[0229] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0230] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0231] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0232] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0233] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0234] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

[0235] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1.

[0236] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1.

[0237] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0238] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD consists of SEQ ID NO: 1.

[0239] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0240] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0241] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to

[0242] TNF.

[0243] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0244] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0245] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0246] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0247] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0248] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0249] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0250] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0251] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0252] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0253] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0254] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating inflammatory bowel disease (IBD) in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0255] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to

[0256] TNF.

[0257] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0258] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0259] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0260] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0261] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0262] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating Crohn's disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0263] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0264] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0265] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0266] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0267] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0268] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to

[0269] TNF.

[0270] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0271] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating ulcerative colitis in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0272] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0273] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0274] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0275] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0276] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0277] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0278] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating celiac disease in a subject, wherein the molecule is administered orally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, and wherein the ISVD binds to TNF.

[0279] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0280] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0281] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0282] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 90% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0283] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 95% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0284] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0285] A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 99% identity to SEQ ID NO: 1, wherein the ISVD binds to TNF, and wherein the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO: 10, and a CDR3 that is the amino acid sequence of SEQ ID NO: 11, wherein the CDRs are defined according to the AbM definition.

[0286] A molecule comprising two immunoglobulin single variable domains (ISVDs) for use in treating a disease in a subject, wherein the first ISVD binds to TNF and has at least 80% identity to SEQ ID NO: 1, wherein the second ISVD binds to TNF and has at least 80% identity to SEQ ID NO: 2, wherein the first and the second ISVD are linked by one or more linkers, such as peptidic linkers, optionally, wherein the molecule comprises one or more additional

[0287] ISVDs. 5.8 Industrial applicability

[0288] The compound for use according to the present invention may be used in the treatment of subjects from a disease, in particular a disease of the Gl tract or a systemic disease.

[0289] 6 Examples

[0290] 6.1 Example 1: Minipig study for determination of ISVD gut stability and permeability

[0291] To analyze the stability and permeability of ISVDs in the gastrointestinal tract of minipigs, two studies were conducted: one using small intestinal (intraduodenal) administration and one using large intestinal (intracecal) administration. Fig. 1 depict a scheme of the experimental setup.

[0292] Methods

[0293] Animal model

[0294] The experiments were carried out using naive adult female Gottingen minipigs (Ellegaard, Soroe Landevej 302, 4261 Dalmose, Denmark) weighing 14.95-16.0 kg (about 6-7 months old) on the day of surgery.

[0295] The animals were housed in social groups of 3 animals (until the surgery) in appropriate kennels (floor area = 4 m2) under standard conditions: room temperature (10-24°C), hygrometry (continuous monitoring), light / dark cycle (12h / 12h, light 8.00 am to 8.00 pm), air replacement (15-20 volumes / hour), water was given ad libitum and food (SDS Dietex, SMP) was given twice a day.

[0296] The animals were allowed to acclimatize to standard environmental conditions for at least

[0297] 10 days prior to surgery. Animals were individually housed after the surgery. Post-surgery, the animals were fed 3 times a day. In addition, the daily ration of food was regularly adapted during the study based on the animals' body weight gain.

[0298] Surgical procedure

[0299] Surgery was performed sequentially in naive female Gottingen minipigs, using the following surgical procedure. Animals were deprived of food in the morning the day before the surgery. The animals were sedated with azaperone before induction of anaesthesia by a mixture of ketamine and xylazine; anaesthesia was maintained with isoflurane. Body temperature was maintained during the surgery using a heating blanket with pulsed air.

[0300] The T-cannula was surgically implanted in minipigs using the surgical method described by Wubben et. al., Contemp Top Lab Anim Sci 40, 27-31 (2001). For placement of the duodenal catheter, connected to a vascular access port (VAP), a median laparotomy was performed, the proximal part of the duodenum was located, and a catheter was inserted over 5 cm into the duodenum in the direction of the distal part of the duodenum and secured; the catheter was cannulated through the abdominal wall and subcutaneously tunnelled up to the neck. A subcutaneous pocket was prepared in the neck and the VAP was connected to the catheter, inserted, and fixed in the pocket. The animals were allowed to recover from surgery for at least 3 weeks before the first administration.

[0301] Administration protocol

[0302] After at least 3 weeks of recovery post-surgery, each minipig received ISDV a total dose of 2g ± 3% diluted in a volume of 20 mL / animal administered via duodenal catheter. Administrations were performed by slow injections over 2 minutes. At the end of intraduodenal administrations, the system VAP + catheter was flushed with 0.9% NaCI with a volume corresponding to the dead volume recorded during the surgery. Different ISDV were administered to the same colony of minipigs with surgically implanted duodenal catheter with at least a period of one week in between two administrations.

[0303] For intracecal administration each minipig received ISDV a total dose of 2g ± 3% diluted in a volume of 20 mL / animal administered via a T-fistula. The ISVDs which were administered via the duodenal catheter are listed in Table 3 below.

[0304] Table 3: ISVDs for intraduodenal administration.

[0305] Intestinal content collection After intraduodenal administration, intestinal content samples were collected via the intestinal T-cannula, at the following time points during the 24 hours post dose: lhr, 2hr, 4hr, 6hr, lOhr and 24hr post administration. Samples were collected into 2 mL screw cap microtubes (Sarstedt) and the net weight of collected sample was measured. Protease inhibitors were added to the collected samples before storing at 70°C until bioanalysis. Feces sample collection

[0306] After each administration ISDV, feces samples were collected post-dose from all individually housed animals at 6h, lOh, 24h, 34h, 48h, 58h and 72h post-administration. All samples were processed in an identical manner. Collected feces samples were homogenized using a BagMixer®. From each processed homogenized feces sample, 3 aliquots (each of 1 mL) were collected in 2 mL screw cap micro tubes (Sarstedt), protease inhibitors were added followed by vortexing and storage at -70°C until bioanalysis. Blood sample collection

[0307] After each ISDV administration, blood samples were collected into dry tubes without anticoagulant (VACUTEST® dry tubes coated with silica micro-particles, without gel, VK054SP, Labelians) from a jugular vein over 24h after each administration: 30 min, lhr, 2hr, 4hr, 6hr, 8hr, lOhr and 24hr post-administration. The blood samples were kept at room temperature for blood clotting, and then centrifuged to collect the serum samples. The separated serum samples were immediately transferred to a freezer (-70°C) in the upright position and were stored until bioanalysis.

[0308] Bioanalysis

[0309] ELISA-based quantification methods were developed and qualified for each ISDV using the target binding assay for quantification of serum, intestinal contents as well as feces homogenates. In those methods, detection was done using electrochemiluminescence (ECL). The LLOQ (lower limit of quantification) for ISDV in serum, intestinal contents and feces homogenate samples was in the range of 1.5 to 13.4 ng / mL, mainly depending on the sample type.

[0310] Results

[0311] Figs. 2 and 3 depict the results for intraduodenal administration of NB1, NB2, NB3 and NB4. Shown is the average ISVD concentration of n=5 (for NB1), n=5 (for NB2), n=4 (for NB3), and n=4 (for NB4) individual minipigs. It can be seen that NB1 is superior to the other 3 tested ISVDs regarding serum concentration (Fig. 2A), ileal concentration (Fig. 2B), and feces concentration (Fig. 3A). Taking into account the logarithmic scale, it is apparent that the concentration of NB1 is several magnitudes higher than the concentration of the other 3 tested ISVDs in all three readouts, especially in the time frame up to 10 h (serum and ileal lumen) and 20 h (feces), respectively. Fig. 3B indicates the total ISVD recovery in feces from feces collected for the period of 72 h post administration. Again, the total recovery for NB1 is higher than for the other 4 ISVDs. Fig. 4 shows the results for intracecal administration of NB1 and NB2. Shown is the average ISVD concentration of 5 individual minipigs for each of NB1 and NB2. It can be seen that NB1 is superior to NB2 regarding serum concentration (Fig. 4A) and feces concentration (Fig. 4B). Taking into account the logarithmic scale, it is apparent that the concentration of NB1 is at least one magnitude higher than the concentration of NB2, especially in the time frame up to 10 h (serum and ileal lumen) and 20 h (feces), respectively.

[0312] To sum it up, it was found that NB1 shows a surprisingly high stability and uptake in serum compared to the other tested ISVDs. This was true for both tested Gl tract administration modes.

[0313] 6.2 Example 2: Rat study for determination of ISVD gut stability and permeability

[0314] To analyze the stability and permeability of ISVDs in another animal model, two studies in rats were conducted: one using small intestinal (intraduodenal) administration and one using large intestinal (intracecal) administration.

[0315] Methods

[0316] Animal model

[0317] Male Wistar Han rats at least of 6 weeks of age were used for this study. Animals underwent surgery for implantation of catheter either in the duodenum (for intra-duodenal administration) or in the cecum (for intracecal administration). Pre- and post-surgery rats were treated with meloxicam and buprenorphine until 72 hours post-surgery. One dose of antibiotic was given during the surgery by intraperitoneal route. Animals were under recovery for at least 5 days post-surgery. Animals were individually housed post-surgery. Animals had access to food (Teklad Global Diets™ (Envigo) 18% Protein Rodent Diet 2018) and tap water ad libitum except during the experiment. Animal quarters were maintained at a temperature between 20°C to 26°C, a relative humidity of 30% to 70%, and with an air flow of at least 10 changes per hour. The light / dark cycle was set for 12 h intervals, but the cycle was interrupted for the performance of study procedures.

[0318] Administration:

[0319] Priorto dosing, the body weight of each animal was recorded. Doses were calculated based on the body weight (kg) and a dose volume of 1 mL / kg. Each rat received ISDV 100 mg / kg body weight (±2 % dose variations). Tested were the ISVDs NB1 and NB2. Intraduodenal and intracecal doses were administered via intraduodenal and intracecal catheters respectively. Immediately after dosing, the catheter was flushed with air (corresponding to dead volume in catheter, i.e. 0.2 mL). Dosing syringes were weighed immediately prior to and immediately after dosing each animal, and the quantity of formulation administered to each animal was determined from the difference in syringe weights. Immediately after dosing, rats were transferred to new cages with bottom mesh without bedding for the collection of excreted feces samples. After collection of the final blood samples, the study animals were euthanized by carbon dioxide asphyxiation in accordance with the American Veterinary Medical Association Guidelines on Euthanasia (Underwood, 2020).

[0320] Blood, Gl content, and feces sample collection and processing

[0321] Blood samples were collected into serum separator tubes. Samples were stored ambient for at least 60 minutes to allow for clotting until processed to serum by centrifugation (5000 rpm at 5°C for 10 minutes). Following centrifugation, serum samples were separated and stored at -70°C until analysis.

[0322] Immediately following terminal blood collections, animals were sacrificed, and the Gl contents of each animal were collected. After excision, the small intestine, cecum and colon were ligated and then dissected to get their contents. Whole contents of each segment were collected into pre-weighted conical tubes by squeezing followed by flushing twice with 500 pL of ice-cold PBS and were collected in the same tube as that of contents. Tubes were vortexed to acquire a homogeneous suspension and immediately snap-frozen with liquid nitrogen and transferred to -70°C until bioanalysis. Excreted feces samples from each animal were collected from their cages into pre-weighted conical tubes and individual sample weights were calculated and recorded for quantification.

[0323] Results

[0324] Fig. 5 depicts the results for intraduodenal administration of NB1 and NB2 in the rat model. Shown is the average ISVD concentration of n=3 for each time point (for NB1) and n=3 for each time point (for NB2) individual rats. Also in the rat model NB1 is superior to NB2 regarding serum concentration (Fig. 5A) and recovery in gut contents and excreted feces (Fig. 5B and C). The serum concentration of NB1 was at least one magnitude higher than the serum concentration of NB2. While recovery of NB1 was over 80% in the first hours after administration, the recovery for NB2 did not exceed 20%.

[0325] Fig. 6 depicts the results for intracecal administration of NB1 and NB2 in the rat model. Shown is the average ISVD concentration of n=3 per time point (for NB1) and n=3 per time point (for NB2) individual rats. As after intraduodenal administration, NB1 showed higher values as NB2 for both serum concentration and recovery, the differences being most pronounced the first hours after administration.

[0326] Therefore, the rat model could confirm the favorable properties of NB1 for administration in the Gl tract.

[0327] 6.3 Example 3: In-vivo efficacy study

[0328] This study investigates the therapeutic effect of ISVDs in ameliorating Dextran Sulfate Sodium (DSS)-induced colitis in transgenic Tgl97 mice overexpressing human tumor necrosis factor (hTNF). Colitis was induced in 6-week-old Tgl97 male mice using 4% DSS in the drinking water for 6 days. From day 6 animals received plain drinking water until the end of the study at day 13. Animals were randomized into four different study groups on day 6 post DSS treatment based on loss of body weight. Two of the study groups received daily oral gavage of ISVDs (NB1 or NB1-NB1) at a dose of 250 mg / kg body weight from day 6 until the end of the study on day 13. For oral administration, the ISVDs were diluted in sodium bicarbonate buffer containing skimmed milk powder (300 mg / mL) and administered via oral gavage. Two other study groups served as negative and positive control, respectively. A phosphate-buffered saline (PBS) treated group served as negative control (i.e. untreated control group). Infliximab, a monoclonal antibody targeting and blocking human TNF, was used as the positive control at a dose of 20 mg / kg body weight. Infliximab was diluted in PBS as vehicle. Both the vehicle (i.e. PBS) and infliximab were administered to the mice via intraperitoneal injections on days 6, 9 and 12, respectively. Body weights for all mice were recorded daily throughout the study. At the end of the study on day 13, blood samples were collected for plasma isolation. Mice were euthanized post blood sample collection on day 13 and colon biopsies were collected for histopathological evaluations.

[0329] NB1-NB1: Molecule comprising an ISVD and an additional ISVD

[0330] The NB1-NB1 molecule is a bivalent molecule comprising two ISVD domains linked by a linker. The sequence of NB1-NB1 is given in SED ID NO: 76. NB1-NB1 consists of the ISVD of SEQ ID NO: 1 and the ISVD of SEQ ID NO: 77 (which is SEQ ID NO: 2 with a terminal Alanine) linked by a linker of seven GGGGS units as given in SEQ ID NO: 78.

[0331] Blood sample processing and mlL6 quantification

[0332] For mlL6 quantification, separated plasma samples were analyzed using the commercially available mlL6 ELISA kit (R&D systems Mouse IL-6 ELISA KIT#M6000B). Plasma samples were analyzed in duplicate and mlL6 levels are reported in pg / mL plasma.

[0333] Histopathological evaluation

[0334] For histopathological evaluation, collected colon tissues were formalin fixed for 24 hours (h) at room temperature and processed using an automatic tissue processor for dehydration, clearing and infiltration of paraffin followed by paraffin embedding. Using a microtome, tissue sections of 4pm were obtained and placed on glass slides. The glass slides with the tissue sections were processed for H / E (hematoxylin & eosin) staining by dipping into hematoxylin solution for 5 min followed by dipping in eosin solution for 1 min with in-between washings. Stained sections were further dehydrated using 100% ethanol followed by clearing with xylene. Finally, a drop of mounting media was added and glass cover slips were fixed on top of tissue sections. An expert pathologist evaluated the stained tissue sections for histopathological damages in a blinded manner based on following three parameters: the inflammation extent, the crypt damage, and the percent involvement. Each of the parameters was assigned a score based on the following observations according to Table 4:

[0335] Table 4: Histopathological scores

[0336] The overall histopathological score for each tissue section was reported as the sum of all three parameters from Table 4 above.

[0337] Results

[0338] Results are shown in Figures 7 to 9. Figure 7 depicts the development in body weight before and after induction of colitis by DSS on day 6. The figure shows that the mice in the vehicle (PBS) treated negative control group had the lowest body weight after colitis induction. In contrast, the positive control group receiving intraperitoneal injections of infliximab on days 6, 9 and 12 demonstrated the highest body weight after colitis induction. Oral administration of the ISVDs NB1 and NB1-NB1 lead to higher body weight than the negative control group after colitis induction.

[0339] Figure 8 depicts the concentration of mouse interleukin 6 (mlL6) in blood plasma at the end of the study on day 13. The figure shows that the negative control group treated with vehicle (PBS) has the highest concentration of mlL6 in the plasma. In contrast, the positive control group treated with infliximab intraperitoneally had the lowest concentration of mlL6 in the plasma. Oral administration of the ISVDs NB1 and NB1-NB1 showed mlL6 concentrations that were lower than the vehicle (PBS) negative control group and comparable to the positive control group receiving injections of infliximab intraperitoneally.

[0340] Figure 9 depicts the aggregated colon histopathology score, i.e. the sum of the extent of inflammation, crypt damage and % involvement collectively. The figure shows that the negative control group treated with vehicle (PBS) has the highest aggregated histopathology score. In contrast, the positive control group treated with infliximab intraperitoneally had the lowest aggregated histopathology score. Oral administration of the ISVDs NB1 and NB1-NB1 showed an aggregated histopathology score that was lower than the negative control and higher than the positive control.

[0341] Figures 7 to 9 of the in-vivo study collectively show that on the day of study termination on day 13 the vehicle (PSB) treated negative control group displayed the lowest body weight in combination with the highest concentration of pro-inflammatory mlL6 in blood plasma and the worst aggregated colon histopathology score. In contrast, the positive control group treated intraperitoneally with Infliximab showed the combination of the highest body weight, the lowest concentration of pro-inflammatory mlL6 in blood plasma and the best aggregated colon histopathology score. Similarto the positive control group, the group receiving oral administration of the ISVDs (NB1 and NB1-NB1) showed the combination of high body weight, low concentration of pro-inflammatory mlL6 in blood plasma and a relatively low aggregated colon histopathology score.

[0342] These findings collectively demonstrate the efficacy of enteral administration of the ISVDs NB1 and NB1-NB1 in ameliorating colitis. Even under challenging administration conditions through oral delivery, the ISVDs maintained efficacy in blocking TNF and leading to reduced inflammation and pathology. The observed efficacy for orally administered ISVDs was relatively lower in comparison to intraperitoneal injections of infliximab. However, it needs to be considered that the oral route of administration is more challenging for the administered molecule due to exposure to intestinal fluids. In addition, NB1 and NB1-NB1 molecules have much shorter half-lives in the blood than infliximab. These factors explain the relatively lower efficacy of the orally administered ISVDs in comparison to intraperitoneally administered infliximab.

[0343] 6.4 Example 4: Evaluation of ISVD stability in pig intestinal fluids

[0344] This study investigates the stability of the ISVDs NB1 and NB1-NB1 in intestinal fluids derived from pigs. The intestinal fluids were collected from farm pigs upon euthanasia from different intestinal compartments, namely duodenum, jejunum and ileum.

[0345] Sample processing and analysis

[0346] For the intestinal stability assessment, the ISVDs (NB1 and NB1-NB1 were incubated in pig derived intestinal fluids separately. Each 100 pl of pig derived fluid was mixed with 100 pl of ISVD solution (0.5 mg / mL) and incubated at 37°C for different time lengths up to 8hr. At different time points, i.e. 0, 2, 4 and 8 hr post incubation, samples were quenched by adding protease inhibitor cocktail and were frozen until further analysis. For the western blot analysis lpl of incubated sample (containing 0.2 pg of ISVD at time point 0) was mixed with 1 pl of NuPAGE sample reducing buffer (10X), 4 pl of nuPAGE 4XLDS sample buffer (Invitrogen NR0008) and 4 pl of PBS. Samples were heated at 98°C for 3 min for denaturation followed by SDS-PAGE (precast 4-12% gel, NuPAGE SDS - Invitrogen NP0321BOX) electrophoresis using IVIES (2-(N-morpholino) ethanesulfonic acid) running buffer. Pre-stained protein ladder (Thermo scientific 26635) was added to one of the wells on each gel as molecular weight marker. Post electrophoresis, blotting was performed to nitrocellulose membrane (Invitrogen IB23001) using iBIot 2TM gel transfer device. The nitrocellulose membranes were blocked using skimmed milk followed by primary antibody incubation. Rabbit (polyclonal) anti-VHH primary antibody was generated in-house, for detection IRDye 680 labelled goat (polyclonal) anti-rabbit IgG (Licor, 92668071) was used. The nitrocellulose membrane was visualized using Oddessy NIR scanner at 700 nm wavelength.

[0347] Results

[0348] The results are shown in Figure 10. Figure 10 depicts Western Blot images of the ISVDs NB1 and NB1-NB1. NB1 is a monovalent VHH with a molecular weight of 12.66 kDa. NB1-NB1 is a bivalent VHH with a molecular weight of 27.46 kDa. The prestained protein ladder (protein marker) contains different proteins ranging from 250 to 11 kDa. Figure 10 shows the results for NB1 on the left and NB1-NB1 on the right. The respective protein bands for each ISVD are indicated by a frame. Figure 10 shows comparable intensities for the NB1 protein bands after 0 hours to 8 hours of incubation in pig intestinal fluids from either jejunum, ileum or duodenum. This demonstrates that NB1 is resistant to proteolytic degradation in pig intestinal fluids for at least 8 hours. Also, the protein bands for NB1-NB1 indicate that the VHH is resistant to proteolytic degradation in pig intestinal fluid for at least 8 hours, especially in intestinal fluids from the ileum and the duodenum.

[0349] 7 References

[0350] Alatab et al., Lancet Gastroenterol Hepatol. 2020 Jan;5(l):17-30.

[0351] Conrath et al., J Biol Chem. 2001 Mar 9;276(10):7346-50

[0352] Davies and Riechmann, FEBS Lett. 1994 Feb 21;339(3):285-90

[0353] Davies and Riechmann, Protein Eng. 1996 Jun;9(6):531-7

[0354] Hamers-Casterman et al., Nature. 1993 Jun 3;363(6428):446-8

[0355] Kabat et al., Sequence of proteins of immunological interest, US Public Health Services, NIH Bethesda, MD, Publication No. 91 Kontermann and Dubel, Eds. 2010, Antibody Engineering, vol 2, Springer Verlag

[0356] Heidelberg Berlin, Martin, Chapter 3, pp. 33-51

[0357] Muyldermans et al. J Biotechnol., 2001 Jun;74(4):277-302

[0358] Underwood, 2020, American Veterinary Medical Association Guidelines on Euthanasia

[0359] Wang et al., BMJ Open. 2023 Mar 28;13(3):e065186

[0360] Wubben et al., Contemp Top Lab Anim Sci. 2001 Nov;40(6):27-31

[0361] Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369)

[0362] Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325-330)

[0363] WO 1994 / 04678

[0364] WO1996 / 34103

[0365] WO1999 / 23221

[0366] W02004 / 041865

[0367] WO2006 / 122787

[0368] W02008 / 020079

[0369] W02012 / 175400

[0370] WO2012 / 175741

[0371] WO2015173325

[0372] W02017 / 080850

[0373] WO2017 / 085172

[0374] WO2018 / 104444

[0375] WO2018 / 134234

[0376] WO2018 / 134235

Claims

69CLAIMS1. A molecule comprising an immunoglobulin single variable domain (ISVD) for use in treating a disease in a subject, wherein the molecule is administered enterally to the subject, wherein the ISVD has at least 80% identity to SEQ ID NO: 1.

2. The molecule for use according to claim 1, wherein the ISVD has at least 90% identity to SEQ ID NO: 1.

3. The molecule for use according to any of the previous claims, wherein the ISVD consists of SEQ ID NO: 1 or SEQ ID NO: 2.

4. The molecule for use according to any of the previous claims, wherein the molecule consists of SEQ ID NO: 1 or SEQ ID NO: 2.

5. The molecule for use according to any of the previous claims, wherein the ISVD binds to tumor necrosis factor (TNF).

6. The molecule for use according to any of the previous claims, where the molecule is administered orally, sublingually, sublabial, buccal, rectally or via an ostomy system to the subject.

7. The molecule for use according to any of the previous claims, where the molecule is administered orally to the subject.

8. The molecule for use according to any of the previous claims, wherein the disease is a systemic disease or a disease of the gastrointestinal tract.

9. The molecule for use according to any of the previous claims, wherein the disease is a disease of the gastrointestinal tract, optionally selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, and infectious diseases of the Gl tract.

10. The molecule for use according to any of claims 1-3 and 5-9, wherein the molecule comprises one or more additional ISVDs.

11. The molecule for use according to claim 10, wherein the ISVD which has at least 80% identity to SEQ ID NO: 1 and the one or more additional ISVDs are linked by one or more linkers, such as peptidic linkers.7012. The molecule for use according to any of claims 10 and 11, wherein the one or more additional ISVDs bind to a target selected from IL-23, IL-22, TNF, alpha4 betal integrin, alpha4 beta? integrin, IL-15, IL-21, IL-6, IL-1, IFN-g, TGF-b, VEGF, Clostridioides difficile toxin A (CdtA), Clostridioides difficile toxin B (CdtB), TL1A, DR5, OX40L, and ICOS.

13. The molecule for use according to any of claims 1-3 and 5-12, wherein the molecule comprises one or more othergroups, residues, moieties, or binding units, optionally linked by one or more linkers, such as peptidic linkers, wherein said one or more other groups, residues, moieties or binding units provide the molecule with increased half-life, increased gut permeability, and / or increased bioavailability, compared to the corresponding molecule without said one or more other groups, residues, moieties or binding units.

14. The molecule for use according to claim 13, wherein said one or more other groups, residues, moieties, or binding units that provide the molecule with increased halflife, increased gut permeability, and / or increased bioavailability are chosen from the group consisting of a. binding units that can bind to serum proteins, b. serum proteins or fragments thereof, c. polyethylene glycol residues, d. binding units that bind to FcRn, e. binding units that bind a receptor for active transport across the gut epithelium, and f. a portion of an active transport domain for transport across the gut epithelium, such as a portion of Cholix protein, and g. fatty acids.

15. A composition comprising a molecule as defined in any of the previous claims for use in treating a disease in a subject, wherein the composition is administered enterally to the subject, wherein the composition comprises the molecule and one or more pharmaceutically acceptable carriers, diluents, excipients and / or71 adjuvants, optionally comprises a protease inhibitor, and optionally comprises one or more further pharmaceutically active compounds.

Images