WO2009080764A2 - Oral or nasal administration of compounds comprising amino acid sequences - Google Patents

Abstract

The present invention relates to compounds comprising amino acid sequences such as single variable domains for oral or nasal administration and pharmaceutical compositions comprising said compounds. More particularly, the invention relates to compounds wherein binding to at least one of the antigens or epitopes selected from the group of epithelial transporters act to increase the bioavailability of the compound in vivo. The present invention furthermore provides various additional methods to enhance bioavailability of said compounds of the invention when using oral or nasal administration.

Description

ORAL OR NASAL ADMINISTRATION OF COMPOUNDS COMPRISING AMINO ACID SEQUENCES

FIELD OF THE INVENTION The present invention relates to compounds comprising amino acid sequences such as single variable domains for oral or nasal (pulmonary) administration and pharmaceutical compositions comprising said compounds. More particularly, the invention relates to compounds wherein binding to at least one of the antigens or epitopes selected from the group of epithelial transporters act to increase the bioavailability of the compound in vivo. The present invention furthermore provides various additional methods to enhance bioavailability of said compounds of the invention over time.

BACKGROUND OF THE INVENTION Administration of conventional low molecular weight drugs by non-invasive routes has been a well established practice. Therapeutic peptides and proteins such as single variable domains and constructs thereof, however, are often unstable, have large molecular weights and are polar in nature. These properties lead to poor permeability through biological membranes. When administered orally, they are susceptible io proteolytic degradation in the gastrointestinal tracts and only pass with difficulty into the body fluids. For this reason, therapeutic peptides and proteins have hitherto been administered mostly by injection, infusion or oral delivery. However, injection, infusion and oral administration are significantly less convenient than, and involve more patient discomfort than, oral administration. Often this inconvenience or discomfort results in substantial patient non- compliance with the treatment regimen. Thus, there is a need in the art for more effective and reproducible oral and/or nasal administration of compounds comprising polypeptides like e.g. single variable domains and/or constructs thereof. Proteolytic enzymes of both the stomach and intestines may degrade polypeptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of polypeptides that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas

(typically having neutral to basic pH optima). Specific difficulties arising from the oral and/or nasal administration of a polypeptide involve the relatively large size of the molecule, and the charge distribution it carries. This may make it more difficult for compound comprising a polypeptide to penetrate the mucus along intestinal walls or to cross the intestinal or pulmonary brush border membrane into the blood.

Oral administration of compounds comprising single variable domains have 2 main challenges to overcome. These challenges are a) degradation by proteolytic enzymes in the stomach and intestine and b) poor absorption, i.e. poor transport of said compounds comprising polypeptides from the apical to the basoϊateral side of the intestine and release into the blood (b) is also an issue for nasal administration. Improving oral effectiveness, i.e. increase of the bioavailability of polypeptidic drugs administrated orally or nasally, is a clear unmet medical need and important for several reasons. First, peptides and proteins are expensive to manufacture either by chemical synthesis or recombinant DNA technologies. Therefore, the more one increases bioavailability, the lesser the amounts that will be required in an oral formulation of a therapeutic drug (economic issue). Second, the greater the bioavailability of a peptide, the less the variability in the dosage absorbed by an individual on a day to day basis (safety issue). Third, the greater the bioavailability of a peptide, the less the concern about breakdown products of the peptide since such breakdown products can act as agonists or antagonists of the receptors where the peptide binds to elicit biological activity (safety issue). Accordingly delivery of therapeutic compounds comprising polypeptides through the oral and/or nasal route receives great attention; it has not been successful and is considered a big hurdle to biological drugs. There is as of today no oral and/or nasal delivery of larger compounds comprising polypeptides (of 100 amino acids and more) approved for human use and there is no established procedure or know how in the art how to formulate a compounds comprising a polypeptide for oral and/or nasal administration, in particular to oral administration.

SUMMARY OF THE INVENTION

The present inventors have now found that a certain class of therapeutic compounds comprising polypeptides such as single variable domains, generally also including peptides but preferably polypeptides that are larger than 100 amino acids in length, can be delivered into the bloodstream by oral administration by using the approaches described herein. The compounds of the invention can be conveniently administered to a subject by the oral route by means of a composition comprising said compounds with the relevant strategies as disclosed herein. Said compounds are characterized and partly shown to be one of the following a) more protease resistant than conventional biologies, e.g. conventional antibodies, b) have typically a higher pH stability or as shown herein (can bind in a pH dependent manner), c) have typically a high temperature stability (i.e. having advantages during processes requiring high T, i.e. in processes of formulation, i.e. compaction and/or granulation), d) have typically a high stability to organic solvents, i.e. may show a superior stability profile to e.g. PLGA solvent exposure (PLGA or poly(lactic-co-glycoiic acid) is an Food and Drug Administration (FDA) approved copolymer which is used in a host of therapeutic devices), e) have shown to have long time stability, f) are typically small globular domains (e.g. in a monovalent form are about 10 times smaller than conventional antibodies) allowing for high loading capacity of matrix or implant, and/or g) have typically high solubility allowing for high loading and highly concentrated doses.

In a first configuration, the present invention provides a further improvement in dual or multi specific compounds comprising polypeptides, e.g. single variable domains, as developed by the present inventors, in which one specificity of the compound is directed towards a protein or polypeptide present in vivo in an organism which can act to transport the compound through the epithelial membrane of the gut by binding to it.

Accordingly, in a first aspect, there is provided a compound comprising an amino acid sequence such as e.g. a single variable domain or nanobody having a binding specificity to a first antigen or epitope and amino acid sequence such as e.g. a single variable domain or nanobody having a binding activity to a second antigen or epitope, wherein one or both of said antigens or epitopes acts to transport the compound through the epithelial membrane into the blood stream in vivo.

Antigens or epitopes which transport the compound from e.g. the gut into the bloodstream as described herein are e.g. present in an organism such as a human. Examples include the Fc receptors, plgR and FcRn, and the VitB12 receptor, preferably plgR and FcRn₅ more preferably FcRn, present in various tissues in the adult organism, e.g. of a human. It is found that plgR, FcRn and the VitB12 receptor may act to increase the concentration of the compounds of the invention in the blood, for example by acting as transporters. Hence, in a second aspect, there is provided a compound as above wherein the antigens or epitopes that act to transport the compound through the epithelial membrane into the blood stream in vivo is selected from the group of pϊgR and FcRn, and the VMS 12 receptor, preferably plgR and FcRn₅ more preferably FcRn.

In another aspect of the invention, the transport across biological membranes can further be improved by amino acid sequences such as single variable domains or nanobodies having a pH dependent binding specificity to a first antigen or epitope and amino acid sequence such as e.g. a single variable domain or nanobody having a binding activity to a second antigen or epitope, wherein said first antigens or epitopes act to transport the compound through the epithelial membrane into the blood stream in vivo and wherein preferably the binding is improved e.g. within the gut. e.g. at acidic pH compared to the binding within the blood stream, e.g. at neutral pH.

Furthermore, the invention provides additional strategies to achieve oral to systemic delivery of compounds comprising amino acid sequences. Such strategies include: a) inhibit proteolytic activity that degrades compounds in stomach and gut, b) develop protease- resistant compound analogs that retain biological activity, c) stabilize the compounds by conjugation to shielding molecules, d) protect the compounds from proteolytic degradation by e.g. enteric coating, e) improve passive transport (diffusion) through the epithelial membrane of the intestine, f) improve active (e.g. receptor mediated or M-cell mediated) trans-epithelial transport of the compounds, and/or g) increase half-life of the compounds in human body, e.g. at target site, for e.g. those active agents that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life.

Moreover, the present invention provides pharmaceutical compositions comprising the compounds of the invention having a bioavailability of the compounds in the range of 1% or more. In one aspect, the lower end of the range is 2%, 3%, 4%₅ 5%, 6%, 7%, 8%, 9% or 10%. In a preferred aspect, orally administered pharmaceutical compositions of the invention are provided that protect the compounds of the invention from proteolytic degradation by an enteric coating. The enteric coating may be a coating known to the skilled person in the art. Moreover, the present invention provides compounds comprising a) a single variable domain directed against the above endogenous transporter proteins, e.g. FcRn, plgR or Vit B12 receptor and b) a single variable domain directed against a target molecule with improved half-life compared to compounds.

Moreover, the neonatal Fc receptor (FcRn) serves to rescue the two most abundant soluble proteins in serum, IgG and albumin, from degradation, and thereby prolong their half- lives (Kim et al, Am J Physiol Gastrointest Liver Physiol, 2006; Ober et ah, Proc Natl Sci, 2004; Ober et ah, J Immunol, 2004). The mechanism is thought to be mediated mainly by endothelial cells that line blood vessels. Inside these cells, FcRn predominantly resides in early/recycling endosomes, where it encounters IgG and albumin internalized by fluid phase endocytosis. The acidic environment of the endosomes facilitates the interaction. Bound IgG and albumin are recycled back to the surface and released from the cell, while unbound ligands are shuttled downstream to lysosomal degradation (Guyer et ah. J Immunol, 1976; Rodewald, J Cell Biol, 1976). While the two ligands bind independently to FcRn, they both do so in a strictly pH-dependent manner, with binding at pH 6.0. but not at pH 7.4 (Chaudhury et ah. Biochem, 2006; Chaudhury et al, J Exp Med, 2003; Ghetie and Ward, Annu Rev Immunol, 2000). The IgG-FcRn interaction is attributed to conserved amino acid residues located at the CH2-CH3 domain interface of IgG Fc with the residues 1253, H310 and H435 as key players (Medesan et ah, J Immunol, 1997). This histidine mediated pH dependency is a result of the imidazole group of histidine. Under endosomal acidic conditions, the group is positively charged and facilitates interaction with negatively charged residues in the FcRn α2-domain, whereas at physiological pH (7.4) the side chain is neutral. The FcRn a2-domain residues involved are E 115, El 16, D 130, Wl 31 and L 135.

Albumin has a number of important and very diverse functions that include the maintenance of the osmotic pressure, buffering capacity, antioxidant action and the transport of a number of substances like fatty acids, bilirubin, hormones, ions and vitamins (Jr., All about albumin: Biochemistry, Genetics, and Medical Applications Academic Press 1996). In addition, as albumin has an unusually long half-life, it is an attractive carrier of therapeutic drugs (Chuang et al., Pharm Res, 2002; Siehle et ah, Anticancer Drugs, 1999: Wunder et ah, J Iimmunol, 2003). The mechanism responsible for the long half life has been given little attention, but recent evidence strongly suggests that FcRn is involved (Chaudhury et al.,

Biochem, 2006; Chaudhury et ah, J Exp Med, 2003; Anderson et ah, Trends Immunol, 2006). Previously, FcRn mediated regulation of the IgG half life has been clearly demonstrated in FcRn-deficient mice (lacking b2m or the FcRn heavy chain) as shown by increased catabolism of IgG compared to normal mice (Ghetie et ah, Eur J Immunol, 1996; Israel et ah, Immunol 1996; Junghans and Anderson, Proc Nat Acad Sci, 1996; Roopenian et ah, J Immunol, 2003). Recently, similar experiments demonstrated that the concentration of albumin was approximately 40 % lower in FcRn heavy chain deficient mice compared with normal mice. Moreover, the data indicate that the capacity of FcRn is extraordinary, as albumin is degraded twice as fast in FcRn-deficient mice as in the WT strain and almost four limes more IgG is saved from degradation than is produced by the mouse (Kim et a!.. Am J Physiol Gastrointest Liver Physiol, 2006). The residues that participate in the interaction between human FcRn (hFcRn) and albumin have not been investigated in detail, however, domain III of human serum albumin (HSA) seems to be involved (Chaudhmy et al.,

Biochem, 2006). The group at UoO has demonstrated that the conserved histidine residue in position 166. located within the α2 domain of the FcRn heavy chain, is critical for the interaction. Guided by the pH dependence of the FcRn-albumin interaction, they compared the sequence of the FcRn α-2 domain from eleven different species, and identified histidine residues that were conserved in all (H 166) or seven (H 161) of these. Both residues are located directly opposite to the IgG interaction site in the folded molecule (see Figure 1). They then did mutagenesis (Hl 61 A) and (Hl 66A) followed by expression and purification of the two FcRn variants as described (Berntzen et aL, J Immunol Meth, 2005). Interaction studies (ELISA and surface plasmon resonance) with these purified receptors and IgG as well as albumin were done to investigate the role of the two histidine residues, and in deed, the results showed clear evidence that the conserved H 166 is a key player in the FcRn-albumin interaction.

Hence, in a preferred embodiment of the invention, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut from apical to basolateral side. In a further preferred embodiment of the invention, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides and shows no signs of immunosuppression, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut from apical to basolateral side and because the FcRn binding unit has a different epitope than IgG or albumin to FcRn no reduction in the half-life of IgGs and albumin are observed. Furthermore, another preferred aspect of this invention are improved FcRn binding units that do have a different epitope on FcRn than IgGs and albumin on FcRn. Those improved FcRn binding units may show an increased half life in mammals, e.g. in humans.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : hFcRn HC binding assay at different pH for a selection of clones (left bar indicates estimate binding at pH 5.1, right bar indicates estimate binding at pH 7.4), Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

Figure 2. Selection of clones binding to shFcRn (panel A) and hFcRn (panel B), but not to b2-microglobulin (panel C) at pH 5.1 (left bar). Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition. Figure 2 also shows a pH dependent binding of the shFcRn nanobodies since the binding at pH 5.1 is decreased by performing washings at neutral pH (pH 7.4, right bar).

Figure 3. shFcRn binding assay at pH 7 for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition. NC-non coated well. Left bars: shFcRN; right bars: b2-microglobulin.

Figure 4. Determination of sliFcRn binding properties of selected Nanobody candidates by ELISA. (A) Binding of the Nanobody candidates to shFcRn-GST at pH 7.4. (B) Binding of the Nanobody candidates to shFcRn-GST at pH 6.0. (C) Binding of serial dilutions of the Nanobody candidates to shFcRn-GST at pH 6.0. (D) Binding of the Nanobody candidates to soluble human β2-microglobulin at pH 6.0. A monoclonal human β2-microglobulin antibody (anti-hp2m mAb) and an anti polyclonal human β2-microglobulin preparation were included as positive controls. The ELISA values represent the mean of duplicates.

Figure 5. Determination of shFcRn binding properties of selected Nanobody candidates by SPR. (A) Binding of the Nanobody candidates to immobilized shFcRn-GST at pH 7.4. (B) Binding of the Nanobody candidates to immobilized shFcRn-GST at pH 6.0.

Figure 6. Influence of of the presence of IgG and HSA on binding of Nanobody candidates to human FcRn. (A) Binding of shFcRn to immobilized MgGl alone or in the absence or presence of Nanobody candidates at pH 6.0. (B) Binding of shFcRn to immobilized HSA alone or in the absence or presence of Nanobody candidates at pH 6.0, The RU values represent the equilibrium binding responses of each injection.

Figure 7. Anti-FcRn Nanobodies as detection reagents. (A) Binding of shFcRn to MgG 1 at pH 6.0 detected with anti-FcRn binding Nanobodies. (B) Binding of sliFcRn to HSA at pH 6.0 detected with anti-FcRn binding Nanobodies. The ELISA values represent the mean of duplicates.

Figure 8. MDCK cells expressing plgR or wildtype MDCK cells were grown in a 12 wells plate. Cells were blocked by addition of Marvel up to a final concentration of 2% Marvel for 1 hour. 3.2 nM of labeled nanobody was allowed to bind to MDCK cells bearing plgR (MDCK+, left bars) or wild type (-) cells (MDCK-, right bars). Binding occurred for 1 hour and cells were washed and lysed before counting. Negative control is 49F5 anti-FcgRI nanobody. The results show that the labeled nanobodies were able to specifically bind to plgR on cells. Labelled IgA was not able to bind.

Figure 9. MDCK ceils expressing plgR were grown in a 12 wells plate. Cells were blocked by addition of Marvel up to a final concentration of 2% Marvel for 1 hour. 3.2 nM of labeled nanobody was allowed to bind to MDCK cells bearing plgR in the absence or presence of 1 uM of unlabeled nanobody. Binding occurred for 1 hour and cells were washed and lysed before counting. Negative control is 49F5 anti-FcgRI nanobody. The results show that the labeled nanobodies were able to specifically bind to plgR on cells, because binding can be competed off with "cold" nanobody. Left bars: labeled nanobodies. Right bars: labeled + unlabeled nanobodies.

Figure 10. 48 or 4.8 nM of nanobody 1D2 and 4B7 and the negative control nanobody was allowed to transcytose over MDCK wildtype and MDCK plgR from basoiateral to apical for 4.5 hours.

Figure 11. Nanobodies 1D2, 1E7, 4B7 and 4Bl 1 were allowed to transcytose over MDCK wildtype and MDCK plgR from basoiateral to apical for 23 hours. Figure 12. Same experiment (with 1D2 and 4B7) as shown in Figure 11 was conducted with IgA present in the basolateral compartment. A 25Ox molar excess of IgA was added to the hasolateral compartment just prior to application of the labeled nanobodies.

Figure 13. Four nanobodies (1D2. 1E7, 4B7 and 4Bl 1) (4.8 nM) were applied apically and samples were taken basolaterally. To avoid re-transcytosis from basolateral back to apical an excess of unlabelled nanobody to the basolateral compartment (marked with +).

Figure 14: Cross-reactivity with mouse plgR in an ELISA. 2 ug/ml mouse or human plgR (R&D) was coated o/n in PBS. 1 uM and 10 nM nanobody was allowed to bind.

Figure 15: Sequence alignments of mouse EpoR-Fc binding nanobodies.

Figure 16: mEpoR-Fc phage binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition. Left bars: mEpor-Fc. Right bars: MgG + Tie2-Fc.

Figure 17: mEpoR-Fc mono and bivalent nanobodies binding assay foi a selection of clones. Negative controls are addition of irrelevant nanobody selected against a viral antigen and no nanobody addition. In each cluster of bars, the order of concentrations and binders from left to right is: ] 0OnM monovalent, 10OnM bivalent, 1OnM monovalent, and 1 OnM bivalent.

Figure 18. mEpo-mEpoR blocking assay of selected P. E. Negative controls are addition of irrelevant P.E. selected against a viral antigen and no P.E. addition. All families, except family V show significant blocking of m£po binding.

Figure 19. mEpo-mEpoR blocking assay of purified nanobodies in a dilution series. Negative controls are addition of irrelevant nanobody selected against a viral antigen and no nanobody addition.

Figure 20. Sequence alignments of GHR binding nanobodies. Figure 21. mGHR-Fc phage binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition. Left bars: mGHR-Fc. Right bars: MgG + Tie2-Fc.

Figure 22. Mono and bivalent nanobodies binding to A- human FCRJI ELISA and B-b2- microglobulin.

Figure 23. Determination of shFcRn binding properties of selected Nanobody candidates by SPR. Binding of Nb candidates at pH 6.0 and pH 7.4: (A) Nb216-E! 1/216-E11 (B) Nb218- Cl/ 218-Cl, (C) Nb218-Gl/ 218-G1, (D) Nb215-E6/215-E6, (E) Nb2ό5-C4/265-C4, (F) Nb265-Eό/265-E6, (G) Nb265-F3/265-F3 and (H) Nb2ό5~E4/265-E4.

Figure 24. Schematic illustrations of two strategies to evaluate monomeric and dimeric versions of the Nb 218-H4. (A) Binding of monomeric and dimeric 218-H5 was measured at pH 6.0 or pH 7.4. The GST-fused shFcRn was immobilized by amine coupling on a CM5 chip. Portions of monomeric or dimeric 218-H4-20GS-218-H4 were injected. (B) Binding of monomeric and dimeric 218-H5 was measured at pH 6.0. Human IgGl was immobilized by amine coupling on a CM5 chip. Portions of monomeric or dimeric 218-H4-20GS-21S-H4 were injected alone or together with monomeric shFcRn (non-GST-fused).

Figure 25 : Binding of monomeric and dimeric Nb variants to shFcRn. (A) Monomeric Nb 218-H5 (100 nM) and monomeric Nb with irrelevant specificity (261-H3; 100 nM) injected at (A) pH 7.4 and (C) pH 6.0. Dimeric Nb 218-H4-20GS-218-H4 (25 nM and 50 nM) and dimeric Nb with irrelevant specificity (261-H3-20GS-261-H3; 25 nM and 50 nM) injected at (A) pH 7.4 and (C) pH 6.0.

Figure 26: Avidity effect of bivalent Nb 218~H4, i.e. SEQ ID NO: 112. (A) Injection of shFcRn, shFcRn together with monomeric Nb218-H4 and shFcRn together with Nb Irrelevant (216-H3) over human IgGl at pH 6.0. (B) Injection of shFcRn, shFcRn together with dimeric 218-H4-20GS-218-H4 and shFcRn together with dimeric Nb Irrelevant (261-H3-20GS-261-H3) over human IgGl at pH 6.0. Figure 27: Kinetic evaluations of Nb 218-H4. (A) A representative sensorgram for binding of different concentrations (500-15 nM) of Nb 218-H4 to immobilized shFcRn. Steady state plots showing the binding responses in resonance units (RU) versus different concentrations ofNb 218-H4 injected over immobilized shFcRn at (B) pH 7.4 and (C) pH 6.0.

Figure 28. Additive binding of Nb 218-H4, human IgGl and HSA to immobilized shFcRn.

(A) Injection of Nb 218-H4. human IgGl and Nb 218-H4/human IgGl at pH 6.0 and pH 7.4.

(B) The equilibrium binding responses (Req) obtained in A. (C) Injection of Nb 218-H4, HSA and Nb 218-H4/HSA at pH 6.0 and pH 7.4. (D) The equilibrium binding responses (Req) obtained in B. (E) Injection of Nb 218-H4, human IgGl/HSA and Nb 218-H4/human IgGl/HSA at pH 6,0 and pH 7.4. (F) The equilibrium binding responses (Req) obtained in E.

Figure 29.¹²³I radiolabelied VHH were able to transcytose over MDCK cells expressing plgR compared to a VHH not binding plgR. All VHH were not able to transcytose over wildtype MDCK cells.

Figure 30.¹²³I radiolabeled VHH binding plgR were able to transcytose over MDCK cells expressing plgR compared to a VHH not binding plgR from apical to baso lateral.

Figure 31. Binding of phages to hpIgR (ELISA). Left bars represent a dilution of 1 μl phage containing solution in 1 ml. Right bars represent a dilution of 10 nl in 1 ml.

Figure 32. Phages were allowed to transcytose for 5 hours. 1D2-phage and 4B7-phage are able to transcytose across the monolayer of MDCK bearing the hpIgR (+), whereas they cannot cross the MDCK cells without hpIgR (-) Also an irrelevant GST-binding phage did not transcytose across transfected or untransfected cells.

Figure 33. mEpoR mono and bivalent nanobodies binding assay for a selection of clones. Negative controls are addition of irrelevant nanobodies (GPA2E6 and GPA2E6_9GS_GPA2E6) selected against a viral antigen. Figure 34. In vivo administration of 4.10- Alb 1 results in weight gain. Body weight changes of C57BL/6 mice injected daily with 4.10-Albl (100 μg/day/mouse; n = 10). Albl (also 100 μg/day /mouse; n = 10) or PBS (n = 10) were followed for 14 days. Data are expressed as average ± SEM (standard error of the mean).

Figure 35. In vivo administration of 4.10- Alb 3 results in increased insulin levels in blood.

Figure 36. 4.10- Albl treatment (100 μg/mouse/day) results in a surge in serum leptin levels after 7 day treatment.

Figure 37. 4.10- Albl aggravates ConA induced hepatitis. Mice were treated daily with 100, 40, and 10 μg/mouse/day or PBS for one week. At day 7, 200 μg ConA was injected i.v. and blood collected at 0, 6 and 24 hours. Serum ALT (panel A) and AST (panel B) are plotted as averages (n = ] 0) ± SEM.

DETAILED DESCRIPTION

The above and other aspects, aspects and advantages of the invention will become clear from the further description herein below.

Definitions: a) By the term "'bioavailability" is meant a term that indicates measurement of total amount of drug that reaches the general circulation from an administered pharmaceutical composition, e.g. from an orally administered pharmaceutical composition, in a single dose or multiple dose setting. It is often expressed in %, i.e. area under the concentration-time curve "AUC" (from 0 time to infinity) or AUC (from o time to 48 or 72 h) of a single dose of the drug when administered e.g. orally, in serum, blood or plasma compared to the AUC (from 0 time to infinity) or AUC (from o time to 48 or 72 h) of single dose of the same amount of drug when injected, i.e. AUC(orally)/AUC(injected) expressed in % (see also "Remington. The Science and Practice of Pharmacy", 21^st Edition, Chapter 53). b) By the term "AUC" is meant area under the concentration-time curve and may be calculated e.g. as defined on page 1042 in Remington, supra). c) By the term "target Molecule" or "target Molecules" or "target" is meant a protein with a biological function in an organism, preferably animal, more preferably mammal most preferred human, wherein said biological function may be involved in the initiation or progression or maintenance of a disease. d) By the term "single variable domain" or "single variable domains" is meant an amino acid sequence or amino acid sequences that forms a single antigen binding unit. Generally, such single variable domains will be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR' s, as further described herein). Such single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. As such, the single variable domain may for example comprise a light chain variable domain sequence (e.g. a V_L~ sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a Vπ-sequence or V_HH 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, as is for example the case for the variable domains that are present in for example conventional antibodies and ScFv fragments that need to interact with another variable domain - e.g. through a V_Π/V_L interaction - to form a functional antigen binding domain). For example, the single variable domain may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), 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™ (as defined herein, and including but not limited to a V_HH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003₅ 21(l l):484-490; as well as to for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629). In particular, the amino acid sequence of the invention may be a Nanobody™ or a suitable fragment thereof. [Note: Nanobody™, Nanobodies™ and Nanoclone ™ are trademarks ofAblynx N. V.] For a further description of V_HH'S and Nanobodies, reference is made to the review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N. V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference, As described in these references, Nanobodies (in particular V_HH sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobodies, including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO07/104529. e) By "high affinity" as used herein is meant a dissociation constant for a monovalent binding Nanobody of (Kd) of < 300 nM and preferably 10 nM and more preferably InM and even more preferably 10OpM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art. f) By "high avidity" as used herein is meant a dissociation constant for a bi- or multivalent binding Nanobody of (Kd) of < 100 nM and preferably 10 nM and more preferably InM and even more preferably lOOpM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art. g) Unless indicated otherwise, the term "immunoglobulin sequence" - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as V_HH domains or V_HA/_L domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence'^", "antibody sequence", '"variable domain sequence", "V_HH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. Also, the term "nucleotide sequence'^* as used herein also encompasses a nucleic acid molecule with said nucleotide sequence, so that the terms "nucleotide sequence'^' and "nucleic acid" should be considered equivalent and are used interchangeably herein. h) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006. 58 (5-6): 640-56: Levin and Weiss, MoI. Biosyst. 2006, 2(1): 49-57; Irving et al, J. Immunol. Methods, 2001. 248(1-2). 31-45; Schraitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol. 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins, i) Amino acid residues will be indicated according to the standard three- letter or one- letter amino acid code, as mentioned in Table A-2;

j) For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity"^' between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity") may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence^'] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings. Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences in accordance with the calculation method outlined heremabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence. Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A- 3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein. Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpoiar or slightly polar residues: Ala, Ser, Thr, Pro and GIy; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpoiar residues: Met, Leu, He, VaI and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala into GIy or into Ser; Arg into Lys; Asn into GIn or into His; Asp into GIu; Cys into Ser; GIn into Asn; GIu into Asp; GIy into Ala or into Pro; His into Asn or into GIn; lie into Leu or into VaI; Leu into lie or into VaI; Lys into Arg, into GIn or into GIu; Met into Leu, into Tyr or into He; Phe into Met. into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into VaL into He or into Leu. Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure. Springer-Verlag. 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. EnzymoL. 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Nad. Acad Sd. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol 157: 105-132. 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V_HH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure. Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional V_H domains form the V_H/V_L interface and potential camelizing substitutions on these positions can be found in the prior art cited above. k) Amino acid sequences and nucleic acid sequences are said to be "exactly the same^"" if they have 100% sequence identity (as defined herein) over their entire length. 1) When comparing two amino acid sequences, the term ''amino acid difference'^' refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences, m) When a nucleotide sequence or amino acid sequence is said to "comprise'^" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a nanobody of the invention is said to comprise a CDR sequence, this may mean that said CDR sequence has been incorporated into the nanobody of the invention, but more usually this generally means that the nanobody of the invention contains within its sequence a stretch of amino acid residues with the same amino acid sequence as said CDR sequence, irrespective of how said nanobody of the invention has been generated or obtained. It should also be noted that when the latter amino acid sequence has a specific biological or structural function, it preferably has essentially the same, a similar or an equivalent biological or structural function in the firstmentioned amino acid sequence (in other words, the firstmentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, a similar or an equivalent biological or structural function). For example, when a nanobody of the invention is said to comprise a CDR sequence or framework sequence, respectively, the CDR sequence and framework are preferably capable, in said nanobody, of functioning as a CDR sequence or framework sequence, respectively. Also, when a nucleotide sequence is said to comprise another nucleotide sequence, the firstmentioned nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the firstmentioned, larger nucleotide sequence), n) A nucleic acid sequence or amino acid sequence is considered to be "(in) essentially isolated (form)^'" - for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered "essentially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid sequence or amino acid sequence that is "in essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis. o) The term "domain" as used herein generally refers to a globular region of an amino acid sequence (such as an antibody chain, and in particular to a globular region of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. Usually, such a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds. The term "binding domain" refers to such a domain that is directed against an antigenic determinant (as defined herein), p) The term "antigenic determinant refers to the epitope on the antigen recognized by the antigen-binding molecule (such as aNanobody or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule. The terms "antigenic determinant" and "epitope" may also be used interchangeably herein, q) An amino acid sequence (such as a nanobody, a compound of the invention, an antibody, a compound of the Invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against or ''''directed against said antigenic determinant, epitope, antigen or protein. r) The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as e.g. a nanobody) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (Kp), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K_D, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K_A), which is 1/K_D). AS will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a nanobody) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as the amino acid sequences, nanobodies and/or single variable domains of the invention) will bind to their antigen with a dissociation constant (K_D) of ICT⁵ to 10^~12 moles/liter or less, and preferably 10^"7 to 10^~12 moles/liter or less and more preferably 10^"8 to 10^-12 moles/liter (i.e. with an association constant (K_A) of 10⁵ to 10¹² liter/ moles or more, and preferably 10⁷ to IQ¹² liter/moles or more and more preferably 10⁸ to 10^!2 liter/moles). Any K_D value greater than 10⁴ mol/liter (or any K_A value lower than I O⁴ M^"s) liters/mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein. The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein, In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10^"4 moles/liter or 10^'3 moles/liter (e.g., of IQ^"2 moles/liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (K_A), by means of the relationship [K_D⁼ 1/K_A]. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the K_D, or dissociation constant, which has units of mol/liler (or M). The affinity can also be expressed as an association constant, K_A, which equals 1/K_D and has units of (mol/Iiter)^-1 (or M^-1). In the present specification, the stability of the interaction between two molecules (such as an amino acid sequence, Nanobody or compound of the invention and its intended target) will mainly be expressed in terms of the K_D value of their interaction; it being clear to the skilled person that in view of the relation K_A =1/K_D, specifying the strength of molecular interaction by its K_D value can also be used to calculate the corresponding K_A value. The K_D- value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG=RT-IH(K_D) (equivalently DG=-RT.1II(KA)), where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm. The K_D for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10^-10M (0.1 nM) to 10^"5M (10000 nM). The stronger an interaction is, the lower is its K_D . The K_D can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as k_off, to the rate of its association, denoted k_on (so that K_D =lWk_cm and K_A⁼ k_on/k_Off). The off-rate k_offhas units s^-1 (where s is the SI unit notation of second). The on-rate k_on has units M^-1S^*1. The on-rate may vary between 10² M^-1S^-1 to about 10⁷ M^-1S^-1, approaching the diffusion-limited association rate constant for biniolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation

, The off-rate may vary between 10^"6 s^-1 (near irreversible complex with a tm of multiple days) to 1 s^-1 (t_1/2=0.69 s). The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et at, Intern. Immunology, 13, 1551-1559. 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k_on, k_Oβ measurements and hence Kø (or K_A) values. This can for example be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_D may correspond to the apparent K_D if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent K_D may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules. Another approach that may be used to assess affinity is the 2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts relating to adsorption of one of the molecules on a support such as plastic. However, the accurate measurement of K_D may be quite labor-intensive and as consequence, often apparent K_D values are determined to assess the binding strength of two molecules. It should be noted that as long all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent K_D measurements can be used as an approximation of the true K_D and hence in the present document K_D and apparent K_D should be treated with equal Importance or relevance. Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecules A and B, one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result an IC₅₀ value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided Ko_ref. the K^ of the reference molecule, is known, as well as the total concentration c_ref of the reference molecule, the apparent K_D for the interaction A-B can be obtained from following formula: K_D

=IC5o/(l+c_ref/ Kp_ref). Note that if c_ref « K_{D re}f_> K_D ~ ΪC50. Provided the measurement of the IC₅₀ is performed in a consistent way (e.g. keeping c_rer fixed) for the binders that are compared, the strength or stability of a molecular interaction can be assessed by the IC₅₀ and this measurement is judged as equivalent to K_D or to apparent K_D throughout this text. s) The half-life of an amino acid sequence, compound or compound of the invention can generally be defined as the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%. in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or compound of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering to a warmblooded animal (i.e. to a human or to another suitable mammal, such as a mouse, rabbit, rat, pig, dog or a primate, for example monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicular is) and/or rhesus monkeys {Macaca mulatto)) and baboon (Papio ur sinus)) a suitable dose of the amino acid sequence, compound or polypeptide of the invention; collecting blood samples or other samples from said animal; determining the level or concentration of the amino acid sequence, compound or compound of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence, compound or compound of the invention has been reduced by 50% compared to the initial level upon dosing. Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth. A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al. Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).

As will also be clear to the skilled person (see for example pages 6 and 7 of WO 04/003019 and in the further references cited therein), the half-life can be expressed using parameters such as the tl/2-alpha, tl/2-beta and the area under the curve (AUC). In the present specification, an "increase in half-life" refers to an increase in any one of these parameters, such as any two of these parameters, or essentially all three these parameters. As used herein "increase in half-life" or "increased half-life" in particular refers to an increase in the tl/2- beta, either with or without an increase in the tl/2-alpha and/or the AUC or both, ϊn the context of the present invention, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of. or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, '"modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved. "Modulating" may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%. such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention. Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or Iigands and/or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions). Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner, t)^: In respect of a target or antigen, the term "interaction site" on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generaliy, an "interaction site" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or compound of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein). u) An amino acid sequence or polypeptide is said to be "specific for" a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a K_D value, K_A value, K_Ofr rate and/or K_on rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a K_D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the K_D with which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen. v) The terms "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target The extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to [target], and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELlSA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target.

The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequence or other binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200- 800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target bindizig sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each ammo acid sequence when passed over the target surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two amino acid sequences are cross-blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-lagged version (R & D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-025). In this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His- tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface. In addition to the example given using N-terminal His-tagged [target], C-terminal His-tagged target could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin). The following generally describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed against a target cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any of the amino acid sequences (or other binding agents such as polypeptides of the invention) described herein. The general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules. The plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target. The amount of bound target is then measured using a reagent that is appropriate to detect the target. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. an Ab-X₅ is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y [target] binding sites per well are at least 10 fold higher than the moles of Ab- X [target] binding sites that were used, per well, during the coating of the ELISA plate, [target] is then added such that the moles of [target] added per well are at least 25-fold lower than the moles of Ab-X [target] binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti- [target] amino acid sequence (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X). second solution phase amino acid sequence (in this case Ab-Y), [target] buffer only (i.e. no target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), target and target detection reagents. The ELISA assay may be run in such a manner so as to have the positive control signal be at least 6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Ab-X and Ab-Y for [target]) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal {i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells). w) As further described herein, the total number of amino acid residues in a nanobody can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein. x) The amino acid residues of a nanobody are numbered according to the general numbering for V_H domains given by Kabat et al. ("Sequence of proteins of immunological interest^"', US Public Health Services, NIH Bethesda, MD₅ Publication No, 91). as applied to V_HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1 -2): 185-195 (see for example Figure 2 of this publication); or referred to herein. According to this numbering, FRl of a nanobody comprises the amino acid residues at positions 1-30. CDRl of a nanobody comprises the amino acid residues at positions 31-35. FR2 of a nanobody comprises the amino acids at positions 36-49, CDR2 of a nanobody comprises the amino acid residues at positions 50-65, FR3 of a nanobody comprises the amino acid residues at positions 66-94, CDR3 of a nanobody comprises the amino acid residues at positions 95-102, and FR4 of a nanobody comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that - as is well known in the art for V_H domains and for V_HH domains - the total number of amino acid residues in each of the CDR^" s may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR's, position 1 according to the Kabat numbering corresponds to the start of FRl and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa]. Alternative methods for numbering the amino acid residues of V_H domains, which methods can also be applied in an analogous manner to V_HH domains from Camelids and to Nanobodies, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, claims and figures, the numbering according to Kabat as applied to V_HH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise. y) An epithelial trans-membrane protein according to the invention is a protein or receptor displayed on the gut membrane which upon binding to a ligancl mediates the transport of said ligand through the membrane, z) The Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicitly indicated otherwise herein.

Strategies to achieve systemic delivery by oral administration of compounds comprising amino acid sequences:

The invention provides one or more of the following main strategies to achieve systemic delivery of orally administered compounds comprising amino acid sequence delivery: a) inhibition of proteolytic activity that degrades polypeptides in stomach and gut, b) developing of protease-resistant polypeptide analogs that retain biological activity, c) stabilizing the polypeptide by conjugation to shielding molecules, d) protecting the polypeptide from proteolytic degradation by e.g. enteric coating, e) improving active (e.g. receptor mediated or M-cell mediated) trans-epithelial transport of the polypeptides, f) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, and/or without being limited to g) improving passive polypeptide transport (diffusion) through the epithelial membrane of the intestine. a) Inhibition of proteolytic activity that degrades polypeptides in stomach and gut The pharmaceutical composition of the invention may comprise agents that inhibit the proteases (i.e. protease inhibitors) present mainly in the stomach but also to a lesser extend in the gut. Such agents are generally known to the skilled person in the art and may be found in e.g. Remington, supra. An example of protease inhibitor is an organic acid such as citric or acetic acid. Protease inhibitors are readily available for the skilled person in the art. b) Development of protease-resistant polypeptide analogs that retain biological activity Hermsen et al. (see Harmsen MM₅ van SoIt CB, van Zijderveld-van Bemmel AM₅ Niewold

TA, van Zijderveld FG. Selection and optimization of proteolyticaliy stable llama single- domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol. 2006 Feb 1; 1-8) showed that stringent selection for proteolytic stability resulted in seven Nanobodies (or VHHs) with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shufjling they further obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These nanobodies or VTTFIs differed by at most ten amino acid residues from each other and were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Similarly- the invention provides pharmaceutical compositions comprising proteolytically stable single variable domains, e.g. Nanobodies or VHHs, wherein said proteolytically stable Nanobodies can be formatted into bi- and/or multivalent (and multimeric) constructs, e.g. into the constructs, polypeptides of the invention. c) Stabilization ϋf the polypeptide by conjugation to shielding molecules It is a further aspect of the invention to provide amino acid sequences which are conjugated to proteolytically "shielding" molecules such as e.g. pegylated polypeptides comprising single variable domains such as e.g. nanobodies and/or dAbs. As mentioned herein, the single variable domains, e.g. nanobodies and/or dAbs and constructs described herein may be pegylated, or contain one or more (additional) amino acid residues that allow for pegylation and/or facilitate pegylation. Two preferred, but non-limiting examples of such polypeptides are TNF55 and TNF56 as described in WO/2006/122786, which both contain an additional cysteine residue for easy attachment of a PEG-group. d) Protection of the polypeptide from proteolytic degradation by e.g. enteric coating Any enteric coating that protects the peptide from stomach proteases and which releases active components of the invention in the intestine is suitable. The enteric coating functions by providing a coating that does not dissolve in low pH environments, such as the stomach. Many enteric coatings are known in the art, and are useful in accordance with the invention. Examples include cellulose acetate phthalate, hydroxypropylmethylethylcellulose succinate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, and methacrylic acid- methyl methacrylate copolymer. It is very desirable that all of the active components be released from the dosage form, and solubilized in the intestinal environment as simultaneously as possible. It is preferred that the dosage form release the active components in the small intestine. e) Improvement of active (e.g. receptor mediated or M-cett mediated) trans-epithelial transport of the polypeptides It is also known that Fc receptors axe involved in transcytosis recycling of proteins and other (biological) molecules. For example, plgR, FcRn, and Vit B 12 receptor is known to be involved in transcytosis through biological membranes such as epithelial layers, e.g. in adult human gut, and FcRn is known to be involved in the recycling of albumin and IgG (see for example Chaudhury et al., The Journal of Experimental Medicine, vol. 3, no. 197, 315-322 (2003)). Thus, the invention provides building blocks, i.e. single variable domains such as nanobodies and/or dAbs binding to plgR, FcRn and/or the Vit B12 receptor. Furthermore, the building block may also be the natural ligand or fragment of ligand. i.e. human Fc part. It is an aspect of the invention to provide pharmaceutical compositions comprising the amino acid sequences of the invention, wherein said amino acid sequences comprise a) at least a single, preferably a bivalent, more preferably a bivalent agonistic, variable domain, e.g. a nanobody, against a target molecule, e.g. human growth hormone (hGH) and/or erythropoietin (EPO), and b) epithelial receptor binding single variable domain (e.g. FcRn, Vit B12 or plgR, preferably FcRn or plgR, more preferably FcRn, binding nanobody).

Compounds comprising amino acid sequences suitable for oral and/or nasal administration and, systemic delivery

The invention relates to compounds comprising an isolated amino acid sequence that is directed against and/or that can specifically bind to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn,

The invention further relates Io compounds comprising an isolated amino acid sequence that is directed against and/or that can specifically bind to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn with a dissociation constant (K_D) of 10^"5 to 10^-12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less.

The invention further relates to compounds comprising an isolated amino acid sequence that is directed against and/or that can specifically bind to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit Bi 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn with a rate of association (k^-rate) of between 10² M⁴S⁴ to about 10⁷ M⁴S⁴, preferably between 10^J M^-1S^-1 and 10⁷ M⁴S^-1, more preferably between 10⁴ M⁴S^-1 and 10⁷ M⁴S⁴, such as between 10⁵ M⁴S⁴ and 10⁷ M⁴S⁴.

The invention further relates to compounds comprising an isolated amino acid sequence that is directed against and/or that can specifically bind to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn₅ even more preferably human FcRn with a rate of dissociation (k_oir rate) between Is⁴ and 10^"6 s⁴, preferably between 10^" s^" and 10^" s^" , more preferably between 10^"3 s⁴ and 10^"6 s⁴, such as between 10^"4 s⁴ and 10^"6 s⁴.

The invention further relates to compounds comprising an isolated amino acid sequence that is directed against and/or that can specifically bind to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The invention further relates to compounds of formula I

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

in which FRl to FR4 refer to amino acid sequences of framework regions 1 to 4 as e.g. described herein for single variable domains, respectively, and in which CDRl Io CDR3 refer to amino acid sequences of the complementarity determining regions 1 to 3 as e.g. described herein for singe variable domains, respectively, and in which optionally X and Y refer to a further unit that comprises one or more other groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers; and wherein said compounds are single variable domains and are directed against a member of the group consisting of plgR, FcRn and V it B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn.

A preferred aspect of the invention relates to the above compounds of formula I, wherein A) CDRl, CDR2 and CDR3 are selected from the group consisting of a) amino acid sequences with SEQ ID NO: 1 to 34 as defined in Table 1, wherein the framework regions are indicated with XXX and wherein CDRl is represented by the first group of amino acid sequence after the first framework region, CDR2 is represented by the second group of amino acid sequence after the second framework region, and CDR3 is represented by the third group of amino acid sequence after the third framework region; or CDRl , CDR2 and CDR3 are selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%. even more preferably 85%. even more preferably 90%. even more preferably 95% identity to the CDRs as shown in SEQ ID NO: 1 to 34 and wherein the amino acid sequences of the framework regions (indicated with XXX) are not taken into account for identity calculation purposes; and wherein optionally

B) said compounds have a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10^" to 10^" moles/litre or less, and preferably 10^-7 to 10^-12 moles/litre or less and more preferably 10^-8 to 10^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and V it Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of between 10² M^-1S^-1 to about 10⁷ M^-1S^-1, preferably between 10³ M^-1S^-1 and 10⁷ M^-1S^-1, more preferably between IG⁴ M^-1S^-1 and 10⁷ M^-1S^-1, such as between IQ⁵ M^-1S^-1 and 10⁷ M^-1S^-1; or said compounds have a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn between 1s^-1 and 10^-6 s- ¹, preferably between 10^-2 s^-1 and 10^"6 s^-1, more preferably between 10^~3 s^-1 and 10^" s^"', such as between 10^"4 S^-1 and 1Q^~6 s^-1 ; or said compounds have an affinity a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM. such as less than 500 pM.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is an immunoglobulin sequence.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a light chain variable domain sequence (e.g. a V_t-sequence); or a heavy chain variable domain sequence (e.g. a V_H- sequence).

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or that is a heavy chain variable domain sequence that is derived from heavy chain antibody.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody (including but not limited to a V_HH sequence).

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a Nanobody.

A preferred aspect of the invention relates to the above compounds of formula I, wherein the amino acid sequence according to any of the preceding aspects is a Nanobody that i) has 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the CDRs of at least one of the amino acid sequences of SEQ ID NO's: 1 to 34 (that are representative for the CDRs), in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the FR sequences are disregarded; and and in which: ii) preferably 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 2; and optionally said compounds have a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10^"5 to 10^-12 moles/litre or less, and preferably 10^"7 to 10^"!2 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting of plgR. FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of between i0² M^"V to about 10⁷ M^-1S^-1, preferably between IG³ M^" V¹ and 10⁷ M^'V^!, more preferably between 10⁴ M^"V^! and 10⁷ MV^!, such as between 10⁵ M⁴S^-1 and 10⁷ 3VfV¹; or said compounds have a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human pϊgR and FcRn, even more preferably human FcRn between 1 s^-1 and 10^"D s^" \ preferably between IQ^'2 s^"J and 10^'6 s^'1, more preferably between 10^"3 s^-1 and IQ^"6 s^-1, such as between 10^"4 s^"! and 10^" s^-1 ; or said compounds have an affinity a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn. even more preferably human FcRn of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM

A preferred aspect of the invention relates to the above compounds of formula I, wherein any of X and Y or both is single variable domain directed against a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker.

A preferred aspect of the invention relates to the above compounds of formula I₅ wherein any of X and Y or both is single variable domain directed against a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker, and wherein examples of single variable domains are provided in Table 3, e.g. amino acid sequence with SEQ ID NOs 69 to 111, and also amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the amino acid sequences of

SEQ ID NO's: 69 to 111. and optionally said compounds have a dissociation constant (K_D) to a member of the group consisting of EPO-R, GH-R. serum albumin or IL-6 receptor, preferably human or mouse EPO-R. GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of 10^"s to 1 O^-12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to IQ^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPQ- R of between 10² M⁴S^-1 to about 10⁷ M^-1S^'1, preferably between 10³ M^-1S^-1 and 10⁷ M^~V, more preferably between \0⁴ M^" V¹ and 10⁷ M⁴S^-1, such as between 10⁵ M^-1S^-1 and 10⁷ M^'V¹; or said compounds have a rate of dissociation (karate) to a member of the group consisting of EPO-R. GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R. GH- R, serum albumin or IL-6 receptor, more preferably human EPO-R₅ GH-R. serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R between Is^-1 and 10^"6 s^-1, preferably between 10^"2 s^-1 and 10^"6 s^~l, more preferably between 10^"3 s^-1 and 10^'6 s^-1, such as between 10^"4 s^-1 and 10^"6 s^'J; or said compounds have an affinity a member of the group consisting of EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The Invention further relates to compounds of formula II

X-Z-Y (II)

in which Z refers to amino acid sequences comprising single variable domains that are directed against a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, and in which optionally X and Y refer to a further unit that comprises one or more other groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers.

A preferred aspect of the invention relates to the above compounds of formula Ii, wherein A) Z refers to amino acid sequences comprising single variable domains that are selected from the group consisting of a) amino acid sequences with SEQ ID NO: 35 to 68 as defined in Table 4; or Z refers to amino acid sequences comprising single variable domains that are selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to amino acid sequences with SEQ ID NO: 35 to 68 as defined in Table 4; and wherein optionally

B) said compounds have a dissociation constant (K_D) to a member of the group consisting of pϊgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10^"5 to 10^" moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn₅ even more preferably human FcRn of between 10² IVF¹S^-1 to about 10⁷ M^-1S^-1, preferably between 10³ M^" V¹ and 10⁷ M^'V¹, more preferably between 10⁴ M^"V^! and 10⁷ IvT¹S^'1. such as between 10⁵ M^'Y¹ and 10⁷ M^~V; or said compounds have a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR. FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn between Is^-1 and 10^"6 s^{" ]}. preferably between 10^~2 s^-1 and 10^"6 s^-1, more preferably between 10^" s^-1 and 10^"6 s^"!, such as between 10^"4 s^-1 and 10^"6 s^"s; or said compounds have an affinity a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

A preferred aspect of the Invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is an immunoglobulin sequence.

A preferred aspect of the invention relates to the above compounds of formula II. wherein the amino acid sequence according to any of the preceding aspects is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi- synthetic immunoglobulin sequence.

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a light chain variable domain sequence (e.g. a Vj_-sequence); or a heavy chain variable domain sequence (e.g. a V_H- sequence).

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a heavy chain variable domain sequence that is derived from a conventional four- chain antibody or that is a heavy chain variable domain sequence that is derived from heavy chain antibody.

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody (including but not limited to a V_HH sequence).

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a Nanobody.

A preferred aspect of the invention relates to the above compounds of formula II, wherein the amino acid sequence according to any of the preceding aspects is a Nanobody that iii) has 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%. even more preferably 95% identity to the amino acid sequences of SEQ ID NO's: 35 to 68, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the FR sequences are disregarded: and and in which: iv) preferably 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 2; and optionally said compounds have a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10° to I O⁴² moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of between IG² M⁴S⁴ to about 10⁷ M⁴S⁴, preferably between 10³ M⁴S⁴ and 10⁷ M^"V\ more preferably between 10⁴ M⁴S⁴ and 10⁷ M^-1S^*1, such as between 10⁵ M⁴S⁴ and 10⁷ M⁴S⁴; or said compounds have a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn between Is⁴ and 10^"6 s^" ', preferably between 10^"2 s⁴ and 10^"6 s^"!, more preferably between 10^"3 s⁴ and 10^"6 s^-1, such as between 10^"4 s^"] and 10^"6 s^"!; or said compounds have an affinity a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of less than 500 iiM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

A preferred aspect of the invention relates to the above compounds of formula II, wherein any of X and Y or both is single variable domain directed against a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker.

A preferred aspect of the invention relates to the above compounds of formula II, wherein any of X and Y or both is single variable domain directed against a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker, and wherein examples of single variable domains are provided in Table 3, e.g. amino acid sequence with SEQ ID NOs 69 to 111, and also amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%. even more preferably 90%, even more preferably 95% identity to the amino acid sequences of SEQ ID NO's: 69 to 111, and optionally said compounds have a dissociation constant (KD) to a member of the group consisting of EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R₅ serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of 10^"5 to 10^-13 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO- R of between 10² VfV¹ to about 10⁷ M⁴S^-1, preferably between 10³ M^'V¹ and 10⁷ M^-1S^'1, more preferably between 10⁴ M⁴S^-1 and 10⁷ M^-1S^-1, such as between 10⁵ M⁴S^-1 and 10⁷ M⁴S^-1; or said compounds have a rate of dissociation (karate) to a member of the group consisting of EPO-R, GH-R₅ serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH- R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R between Is^" and 10^" s^" , preferably between IG^"2 s^-1 and 10^"6 more preferably between 10^*3 s^-1 and 10^"6 s^-1, such as between IG^"4 s^-1 and 10^"6 s⁴; or said compounds have an affinity a member of the group consisting of EPO-R, GH-R. serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R. GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

In an aspect, the compounds of the invention comprising e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, may also form a sequence or signal that allows said Compounds of the invention comprising e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, to be directed towards and/or to penetrate or enter into specific gut epithelial cells, or parts or compartments of said cells, and/or that allows the Compounds of the invention comprising e.g. at least one Nanobody and/or dAb, preferably a Nanobody, to penetrate or cross a biological barrier such as the gut wall or membrane.

In another preferred aspect the Compounds or construct of the Invention is a multispecific polypeptide comprising at least one Nanobody, domain antibody, single domain antibody or dAb directed against a target and at least one Nanobody, domain antibody, single domain antibody or dAb that directs the compound of the invention towards, and/or that allows the compound of the invention to penetrate or to enter into specific gut membrane cells, or parts or compartments of said cells, and/or that allows the Compound of the invention to penetrate or cross a biological barrier such as the gut wall or a cell layer of said wall, e.g. membrane. Examples of such Nanobodies, domain antibodies, single domain antibodies or dAbs include Nanobodies, domain antibodies, single domain antibodies or dAbs that are directed towards specific cell-surface proteins, receptors, markers or epitopes of the gut membrane cells.

In this context, the Compounds of the invention comprising e.g. at least one Nanobody and/or dAb. preferably a Nanobody, may comprise one or more Nanobodies, domain antibodies, single domain antibodies or dAbs directed against the desired target and one or more ligand (also called membrane crossing ligand) directed against an epithelial trans-membrane protein on the mucosal membrane, wherein said polypeptide crosses the mucosal membrane upon binding of the ligand to said epithelial trans-membrane protein. In one aspect of the present invention, the ligand is a Compound of the Invention, e.g. a single variable domain, a Nanobαdy, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine. The polypeptide or protein crosses the wall upon binding of said Nanobody, domain antibody, single domain antibody or dAb to said epithelial trans-membrane protein. The membrane crossing Nanobody, domain antibody, single domain antibody or dAb may be prepared from a peptide library which is screened for binding to the epithelial transmembrane protein or for crossing properties. Examples of such single variable domains, e.g. Nanobodies, directed against said epithelial trans-membrane protein are the Nanobodies against FcRn. plgR and/or VitB 12 receptor as disclosed in the experimental part.

In another aspect, the Compounds of the invention comprise e.g. at least one single variable domain, a Nanobody and/or a dAb. preferably a Nanobody, and in addition a therapeutic polypeptide or agent, e.g. a Compound of the Invention, e.g. against a Target Molecule, which is covalently or non-covalently linked to said single variable domain, Nanobody, domain antibody, single domain antibody or dAb that is directed against an epithelial transmembrane protein on the gut membrane. It is an aspect of the invention that these single variable domains, Nanobodies, domain antibodies, single domain antibodies or dAbs can be added as a tag to Compounds of the invention comprising e.g. at least one Nanobody and/or a dAb, preferably a Nanobody. for crossing or passage through the epithelial membrane. Examples of such a therapeutic polypeptide or agent are Nanobodies against FcRn. plgR and/or VitB 12 receptor.

In yet another aspect, the Compounds of the invention comprise e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, directed against the desired Target Molecule and another ligand (e.g. a natural ligand) of the epithelial trans-membrane protein. The resulting Polypeptide, upon binding of the ligand to the epithelial trans-membrane protein, is transported through the membrane. An example of such ligand (e.g. a natural ligand) of the epithelial trans-membrane protein is the Fc unit or fragment thereof of a human antibody, e.g. the Fc unit of human IgGl .

In yet another aspect of the present invention, the ligand is a Compound of the Invention, e.g. a polypeptide comprising a single variable domain, a Nanobody, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine, and wherein said Compound of the Invention, e.g. a single variable domain, a Nanobody, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, binds to said tranε- membrane protein In a pH dependent manner, preferably binds better at acidic pH, e.g. pH 7 or less, e.g. pH5 or pH6, than at neutral physiological pH such as pH 7 or more, e.g. pH 7.4. Such pH dependent single variable domains, e.g. Nanobodies, are exemplified In this application (pH dependent human FcRn and pH dependent human serum albumin binders) and are disclosed in the experimental part.

In yet another aspect, the Compounds of the invention comprise e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, directed against the desired Target Molecule and at least another single variable domain, e.g. Nanobody, domain antibody, single domain antibody or dAb that is directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine, and wherein said other single variable domain, e.g. Nanobody, domain antibody, single domain antibody or dAb binds to said trans-membrane protein in a pH dependent manner, preferably binds better at acidic pH. e.g. pH 7 or less, e.g. pH 5 or pH 6, than at neutral physiological pH such as pH 7 or more, e.g. pH 7.4, The resulting Polypeptide, upon binding of the ligand to the epithelial trans-membrane protein, is transported through the membrane. An example of such ligand (e.g. a natural ligand) of the epithelial trans-membrane protein is the Fc unit or fragment thereof of a human antibody, e.g. the Fc unit of human IgGl.

Particular aspects of the invention relate to compounds of formula I or II and are hereinafter also referred to as Compounds of the invention.

Alternative aspects:

> Increase of half-life of the compoμnd of the invention in human body, e.g. at target site. for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by cpyalently binding an unit allowing for longer half life In one specific aspect of the invention, a Compound of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such Compounds of the invention will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or amino acid sequences that is linked to at least one moiety that increases the half-life of the Polypeptide of the Invention (e.g. FcRn binding unit that does not bind to IgG or albumin binding site of FcRn). Examples of Compounds of the invention that comprise such half- life extending moieties or amino acid sequences are clear to the skilled person; and for example include, without limitation, polypeptides in which the one or more amino acid sequences are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, single variable domains such as domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"^"s, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrine; reference is made to the further description and references mentioned herein, see e.g. also WO 2007/112940); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/017 '43, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. filed on December 5, 2006 (see also PCT/EP2007/063348). Generally, the compounds of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se. In a preferred, but non-limiting aspect of the invention, such Compounds of the invention has a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se. In another preferred, but non-limiting aspect of the invention, such Compounds of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, Compounds of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 1 1 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

> Improyement of passive polypeptide, .transport (diffusion) through, the epithelial membrane of the intestine

The Compositions of the Invention may further comprise one or more permeation enhancer. As used herein, trans-epithelial permeation enhancers include agents which enhance the release or solubility (e.g., from a formulation delivery vehicle), diffusion rate, penetration capacity and timing, uptake, residence time, stability, effective half-life, peak or sustained concentration levels, clearance and other desired delivery characteristics (e.g. as measured at the site of delivery, or at a selected target site of activity such as the bloodstream and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain) of the Compounds of the invention or of additional biologically active ingredient(s). Enhancement of passive transport through intestinal gut wall can thus occur by any of a variety of relevant mechanisms, for example by increasing the diffusion, increasing membrane fluidity, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing mucosal membrane components (e.g. lipids), changing non-protein and protein sulfhydryl levels in epithelial tissues, increasing water flux across the surface, modulating epithelial junctional physiology, reducing the viscosity of mucus overlying the epithelium, reducing mucociliary clearance rates, increasing blood flow and other mechanisms. Suitable permeability enhancing agents will be clear to a person skilled in the art of pharmacology and are further described hereafter. Such agents may be used in suitable amounts known per se, which will be clear to the skilled person based on the disclosure and prior art cited herein.

Permeability enhancing agents include (a) aggregation inhibitory agents, (b) charge modifying agents, (c) mucolytic or mucus clearing agents, (d) ciliostatic agents; (f) membrane penetration-enhancing agents such as acylcarnitine, (g) modulatory agents of epithelial junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (h) vasodilator agents, and (i) stabilizing delivery vehicles, carriers, supports or complex- forming species with which the Compound of the invention is effectively combined. associated, contained, encapsulated or bound to stabilize the Compound of the invention for enhanced intestinal transport. These agents are further exemplified - without being limiting as additional agents comprised in the compositions of the present invention - in, WO98034632, WO9736480 and/or WO9630036.

In a further aspect, a membrane penetration-enhancing agent is added to the composition of the present invention. Different membrane penetration-enhancing agents have been described such as (i) a surfactant, (ii) a bile salt or bile salt derivative, (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enarnine, (vi) an NO donor compound, (vii) a long-chain amphipathic molecule (viii) a small hydrophobic penetration enhancer, (ix) sodium or a salicylic acid derivative, (x) a glycerol ester of acetoacetic acid, (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) a chelating agent (e.g., citric acid, salicylates), (xiv) an amino acid or salt thereof, (xv) an N-acetyϊamino acid or salt thereof, (xvi) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis, (xix) cationic polymers, or (xx) any combination of the membrane penetration enhancing agents of ((i)-(xix)). The membrane penetration-enhancing agent can be selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Alternatively, long-chain amphipathic molecules, for example, deacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and the bile salts (e.g., unsaturated cyclic ureas and Transcutol), may be employed to enhance mucosal penetration of the Nanobodies, polypeptides or proteins of the invention, ϊn additional aspects, surfactants (e.g., Tween 80, Poloxamer 188, polysorbates; further non-limiting examples of surfactants are also provided in EP 490806, US 5,759,565, and WO 04/093917) are employed as adjunct compounds, processing agents, or formulation additives to enhance oral delivery of the Nanobodies, polypeptides or proteins of the invention. These penetration-enhancing agents typically interact at either the polar head groups or the hydrophilic tail regions of molecules that comprise the lipid bilayer of epithelial cells lining the oral mucosa (Barry, Pharmacology of the Skin, Vol. 1, pp. 121-137, Shroot et al, Eds., Karger, Basel 1987; and Barry, J. Controlled Release 1987; 6: 85-97). Interaction at these sites may have the effect of disrupting the packing of the lipid molecules, increasing the fluidity of the bilayer, and facilitating transport of the Compounds of the invention across the mucosal barrier. Additional non-limiting examples of membrane penetration-enhancing agent are described in WO 04/093917, WO 05/120551 and Davis and Ilium (Clin. Pharmacokinet 2003, 42: 1107- 1128).

In various aspects of the invention, the Compound of the invention is combined with one, two, three, four or more of the permeability enhancing agents recited in (a)-(k) above. These agents may be admixed, alone or together, with the oral carrier and with the Compound of the Invention, or otherwise combined therewith in a pharmaceutically acceptable formulation or delivery vehicle.

While the mechanism of absorption promotion may vary with different permeability - enhancing agents of the invention, useful reagents in this context will not substantially adversely affect the tissue and will be selected according to the physico chemical characteristics of the particular Compound of the invention or other active ingredients or deliver}' enhancing agent. In this context, delivery-enhancing agents that increase penetration or permeability of the gut wall will often result in some alteration of the protective permeability barrier of the gut. For such delivery- enhancing agents to be of value within the invention, it is generally desired that any significant changes in permeability of the gut be reversible within a time frame appropriate to the desired duration of drug delivery. Furthermore, there should be no substantial, cumulative toxicity, nor any permanent deleterious changes induced in the barrier properties of the gut with long term use.

Some preferred aspects are using the above disclosed strategies are provided below: Enteric coating:

In one of the aspects of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, more preferably agonistic polypeptides, systemic and/or local (i.e. topical gut) delivery, is provided through oral administration by protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the ait; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half- life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of a suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder. In a preferred aspect, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn₅ more preferably FcRn mediated trans-epithelial transport.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM. more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3. 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn₅ more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder. In a preferred aspect, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) develop protease-resistant polypeptide analogs that retain biological activity, e.g. pharmaceutical oral compositions comprising Target Molecule binding single variable domains, e.g. Nanobodies or dAbs, selected for protease resistance by at least 2, 3, 4. 5 10, 20, 50 100 folds (see e.g. experimental part); c) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and d) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder. In a preferred aspect, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM. preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and [c) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or d) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcaraitine and/or Eligen® carrier technology]. In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport: and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder; and [d) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or e) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcamitine and/or Eligeπ® carrier technology].

In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and [c) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or d) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcamitine and/or Eligen® carrier technology]. In another aspect of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) providing continuous local (topical in gut) delivery by bacterial system, e.g. lactic acid bacteria.

Further aspects:

Method of selection

Another aspect of the present invention is a method for selecting Nanobodies, domain antibodies, single domain antibodies or dAbs directed against an epithelial trans-membrane protein, wherein said Nanobody, domain antibody, single domain antibody or dAb crosses the gut membrane upon binding to said epithelial trans-membrane protein. Said method comprises panning epithelial trans-membrane protein-displaying membranes with a phage library (naϊve or immune) of Nanobodies, domain antibodies, single domain antibodies or dAbs. and selecting for membrane crossing Nanobodies, domain antibodies, single domain antibodies or dAbs by recovering the transported phage from the membrane. The invention includes a selection method which uses cell lines that over-expresses an epithelial transmembrane protein or cell lines transfected with an epithelial trans-membrane protein gene to allow the easy selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs binding to the epithelial trans-membrane protein. This avoids the need for protein expression and purification, speeding up significantly the generation of membrane crossing Nanobodies, domain antibodies, single domain antibodies or dAbs.

In another aspect, the invention includes a selection method using cells to allow the selection of phage single variable domains, Nanobodies, domain antibodies, single domain antibodies or dAbs that show receptor mediated internalization. Said method comprises adding the phage Nanobodies. domain antibodies, single domain antibodies or dAbs to the cells and recovering the phage Nanobodies, domain antibodies, single domain antibodies or dAbs from the cells that have undergone internalization, In yet another aspect, the invention includes a selection method using cells seeded on a filter or in a Transwell system or Boyden chamber to allow the selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs that transcytose through the cell monolayer. Said method comprises adding the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to compartment 1, allow the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to migrate across the cell monolayer and harvest the phage Nanobodies that migrate in compartment 2. Alternatively, the polypeptides of the invention comprising e.g. at least a Nanobody or a dAb against a target molecule, may also be suitably formulated per se for oral delivery e.g. in the form of a powder (such as a freeze-dried or micronized powder) or mist.

Pharmaceutical Composition

Moreover, in one aspect anti- aggregation agents are added Io the composition of the invention. Aggregation inhibitory agents include, for example, polymers of various functionalities, such as polyethylene glycol, dextran, diethyl aminoethyl dextran, and carboxymethyi cellulose, which significantly increase the stability and reduce the solid-phase aggregation of polypeptides admixed therewith or linked thereto. In some instances, the activity or physical stability of polypeptides can also be enhanced by various additives to pharmaceutical compositions comprising the Polypeptide of the Invention. For example, additives, such as polyols (including sugars), amino acids, and various salts may be used. Certain additives, in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized polypeptides. These additives can also be used within the invention to protect the polypeptides against aggregation not only during lyophilization but also during storage in the dry state. For example, sucrose and Ficoil 70 (a polymer with sucrose units) exhibit significant protection against polypeptide aggregation during solid- phase incubation under various conditions. These additives may also enhance the stability of solid polypeptides embedded within polymer matrices. Yet additional additives, for example sucrose, stabilize polypeptides against solid-state aggregation in humid atmospheres at elevated temperatures, as may occur in certain sustained-release formulations of the invention. These additives can be incorporated into polymeric melt processes and compositions within the invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing various stabilizing additives described above. Sustained release of unaggregated polypeptides can thereby be obtained over an extended period of time. A wide non-limiting range of suitable methods and anti- aggregation agents are available for incorporation within the compositions of the invention such as disclosed in WO 05/120551. Breslow et al. (J. Am. Chem. Soc. 1996;! 18: 11678- 11681), Breslow et al.(PNAS USA 1997; 94: 11156-11158), Breslow et al. (Tetrahedron Lett. 1998; 2887-2890), Zutshi et al. (Curr. Opin. Chem. Biol. 1998; 2: 62-66), Daugherty et al. (J. Am. Chem. Soc. 1999; 121 : 4325-4333), Zutshi et al. (J. Am. Chem. Soc. 1997; 119: 4841- 4845), Ghosh et al. (Chem. Biol. 1997; 5: 439-445), Hamuro et al. (Angew. Chem. Int. Ed. Engl. 1997; 36: 2680-2683), Alberg et al., Science 1993; 262: 248-250), Tauton et al. (J. Am. Chem. Soc. 1996; 118: 10412-10422), Park et al. (J. Am. Chem. Soc. 1999; 121 : 843), Prasarrna et al. (Biochemistry 1998; 37:6883-6893), Tiley et al. (J. Am. Chem. Soc. 1997; 119: 7589-7590), Judice et al. (PNAS USA 1997; 94: 1342643430), Fan et al. (J. Am. Chem. Soc. 1998; 120: 8893-8894), Gamboni et al. (Biochemistry 1998; 37: 1218942194).

In another aspect, enzyme inhibitors are added to the composition of the invention. The stomach and gut contain hydrolytic enzymes, such as lipases and proteases, which must be overcome. This enzymatic "barrier" can be dampened by administering enzyme inhibitors that prevent or at least lessen the extent of degradation. Enzyme inhibitors for use within the invention are selected from a wide range of non-protein inhibitors that vary in their degree of potency and toxicity (see, e.g., L. Stryer, Biochemistry, WH: Freeman and Company, NY. NY, 1988). Non-limiting examples include amastatin and bestatin (O'Hagan et al., Pbarm. Res, 1990, 7: 772-776). Various classes of enzyme inhibitors are extensively described and exemplified in WO 05/120551 without being limiting for use in the composition of the present invention. Another means to inhibit degradation is pegyiation with PEG molecules, preferably low molecular weight PEG molecules (e.g. 2 kDa; Lee et al., Calcif Tissue Int. 2003, 73: 545-549). Also within the scope of the present invention is the use, as enzyme inhibitor, of a Nanobody, domain antibody, single domain antibody or "dAb" directed against said enzyme. Accordingly, the invention also relates to a bispecific or multispecific Polypeptide comprising or essentially consisting of one or more Nanobodies, domain antibodies, single domain antibodies or "dAbs" directed against the desired target and one or more Nanobodies, domain antibodies, single domain antibodies or "dAbs" directed against an enzyme of the stomach and/or gut.

In addition to the Compound of the invention and, optionally, one or more additives and/or agents, the composition of the invention may further comprise one or more additional therapeutic ingredients (or active substances). These therapeutic ingredients can be any compound that elicits a desired activity or therapeutic or biological response in the subject. In a preferred aspect, two or more Nanobodies the invention may be used in combination, i.e. as a combined treatment regimen. As indicated above, the pharmaceutical composition of the invention should comprise at least a therapeutically effective amount of the Compound of the Invention, e.g. the polypeptides comprising single variable domains, e.g. Nanobodies. A "therapeutically effective amount" as used in the present invention in its broadest sense means an amount of the Compound of the invention that is capable of eliciting the desired activity or the desired biological, prophylactic and/or therapeutic response. The amount of Compound of the invention to be administered and hence the amount of active ingredient in the pharmaceutical composition of the invention will, of course, vary according to factors such as the bioavailability of the polypeptide, the disease indication and particular status of the subject (e.g., the subject's age, size, fitness, extent of symptoms, susceptibility factors, etc), the target cell, tumor, tissue, graft or organ, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the Compounds of the invention for eliciting the desired activity or biological, prophylactic or therapeutic response in the subject. Dosage regimens may be adjusted to provide an optimum activity or biological, prophylactic or therapeutic response. Dosages should also be adjusted based on the release rate of the administered formulation (e.g. a slow release polymer containing composition versus a capsule comprising pressed Polypeptide of the Invention). A therapeutically effective amount is also one in which any toxic or detrimental side effects of the Compound of the invention are outweighed in clinical terms by therapeutically beneficial effects. Doses may be chosen to be equipotent to the injection route.

In this context, the absolute bioavailability of the Compound of the invention following oral administration of the Pharmaceutical Composition of the Invention is of the order of ca. 1, 2, 3, 5, 7, 10, 15, 20, 25. 30, 40, 50, 60, 70, 80, 90, 100% or more of the levels achieved with the corresponding injection. Absolute bioavailability measures the availability of the active drug in systemic circulation after oral administration when compared with intravenous administration. The absolute bioavailability of the Compounds of the invention is determined by comparing the concentration vs. time plot of the Compounds of the invention after intravenous (IV) administration with the concentration vs. time plot of the Compounds of the invention after oral (IN) administration. The absolute bioavailability of Compounds of the invention is also defined as (AUC_IN X doseiN)/(AUCiv x doseiv) x 100. The relative bioavailability of the Compounds of the invention following oral administration of the Pharmaceutical Composition of the Invention is of the order of ca. 1, 2, 3, 5. 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100% or more of the levels achieved with the corresponding injection. Relative bioavailability measures the availability of the active drug in systemic circulation after oral administration when compared with another form of administration of the same drug, such as intramuscular (IM) or subcutaneous (SC). The relative bioavailability of Compounds of the invention is determined by comparing the concentration vs. time plot of Compounds of the invention after intramuscular (IM) or subcutaneous (SC) administration with the concentration vs. time plot of Compounds of the invention after oral (IN) administration. The relative bioavailability of Compounds of the invention is defined as (AUCIN X doseiN)/(AUCsc/iM x dosesc/iM) x 100. Accordingly, in order to be equipotent to the injection route, oral administration will appropriately be effected so as to give a dosage rate of the order of 1 to 100 times, preferably 1 to 50 times, more preferably 1 to 20 times, even more preferably 1 to 10 times the dosage required for treatment via injection, also depending on the frequency of the oral application.

The amount of active compound will generally be chosen to provide effective treatment on administration once a day or once a week or once a month. Alternatively, dosages may be split over a series of e.g. 1 to 4 applications taken at intervals during the day, week or month. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple takings of a plurality of pills or capsules. To maintain more consistent or normalized therapeutic levels of the Compound of the Invention, it may be advisable that the Composition of the Invention is repeatedly administered to the subject, for example one, two or more times within a 24 hour period, four or more times within a 24 hour period, six or more times within a 24 hour period, or eight or more times within a 24 hour period. An administration regimen could include long-term, daily, weekly or monthly treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. The clinician will generally be able to determine a suitable daily, weekly or monthly dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. The final determination of the effective dosage will be based on animal model studies, followed up by human clinical trials, and is guided by determining effective dosages and oral administration protocols that significantly reduce the occurrence or severity of the targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Ultimately, the dosage of Compounds of the invention will be at the discretion of the attendant, physician or clinician. The dosage can also be adjusted by the individual physician in the event of any complication.

As a non-limiting example, the Compounds of the invention is suitably presented in the Pharmaceutical Composition of the Invention in an amount such as to provide a free Compounds of the invention concentration from about 0.1 microgram to 0.1 gram per kg body weight per day, such as from 1 microgram to 0.1 gram per kg body weight per day, such as from 0.01 to 100 milligram per kg body weight per day, such as from 0.05-100 milligram, such as from 0.05 to 50 milligram. 0.05 to 30 milligram, 0.1 to 20 milligram, or from about 1 to J 0 or about 5 to 10 milligram per kg body weight per day either as a single daily dose or as multiple divided doses during the day.

The proportion of each further component in the oral composition of the invention may vary depending on the components used. For example, but without being limiting, the amount of enteric coating may be in the range of from 0.1 to 99.9 %, preferably 1 to 20% by weight of the total weight of the composition. When present, the amount of permeability enhancer may be in the range from about 0.03 to about 10% or higher and preferably about 0.05 to about 1.0% by weight of the total weight of the composition, the amount depending on the specific enhancer used. The amount is generally kept as low as possible since above a certain level no further enhancement of absorption can be achieved and also too high of a enhancer level may cause irritation of the gut. The amount of protease inhibitor may be at least 0.1%, suitably in the range from about 0.5 to 10 % of the total weight of the composition. Preserving agents may be present in an amount of from about 0.002 to 0.02 % by weight of the total weight or volume of the composition. The amount of the other excipients will be determined by processes known to the skilled person in the art. In addition to the concentration of the different compounds in the composition of the invention, the total delivery weight is important to consider as well. The delivery weight is relatively high for oral compositions and may be up to 1 g or more. Suitable delivery weights will be clear to a person skilled in the art of pharmacology.

Method of preparation of pharmaceutical composition:

The present invention further provides a method for the preparation of a composition mixing the Polypeptides of the Invention, e.g. the single variable domains, Nanobodies, the domain antibodies, the single domain antibodies or the dAbs and the pharmaceutically acceptable excipients (as proposed herein, e.g. protease inhibitors, slow release matrices, and/or permeability enhancer) and thus resulting in a powder that is then further e.g. filled into capsules, preferably enterically coated capsules. Alternatively, said powder comprising the Compounds of the invention and the excipients are milled into smaller granules (dry or wet granulation) and pressed into the core pill - said core pill is then further coated e.g. by enteric coating. All above described steps may be prepared in a conventional manner known to the skilled person in pharmacology.

The solid oral composition of the invention may be prepared in conventional manner. For example, the Polypeptides of the Invention, e.g. Nanobodies, may be admixed with the protease inhibitors, slow release matrices, and/or permeability enhancer, optionally with further ingredients, additives and/or agents as indicated above. The Polypeptides of the Invention, e.g. Nanobodies, may be in solution e.g. an aqueous or alcoholic solution when being mixed with the protease inhibitors, slow release matrices, and/or permeability enhancer and the solvent evaporated, e.g. under freeze-drying or spray drying. Such drying may be effected under the conventional conditions. Alternatively the dry mixtures may be compacted and/or granulated and then be pulverized and/or sieved. If desired the compacted composition may be further coated. According to a preferred aspect of the invention, the oral composition is prepared by lyophilisation, then granulated and filled up into enterically coated capsules. A homogeneous solution, preferably aqueous, containing the Polypeptides of the Invention, e.g. Nanobodies, and optionally containing further ingredients, additives and/or agents as discussed above, e.g. protease inhibitors, slow release matrices, and/or permeability enhancer, is prepared and then submitted to lyophilisation in analogy with known lyophilisation procedures, and to subsequent drying. The resulting powder may then be filled up into enterically coaled capsules before administration.

Alternatively, the Compounds of the invention may be administered in liquid form such as in the form of a suspension or partly or fully dissolved solution, e.g. the lyophilized powder may be reconstituted in e.g. water before administration or may be stored in liquid form and thus may be directly be used as such.

For administration of a liquid, for example, such compositions will suitably be put up in a container provided with a conventional dropper/closure device, e.g. comprising a pipette or the like, preferably delivering a substantially fixed volume of composition/drop.

If desired a powder or liquid may be filled into a soft or hard capsule adapted for oral administration. The powder may be sieved before filled into the capsules such as gelatine capsules, preferably an enterically coated capsule.

The Pharmaceutical Composition of the Invention is formulated for oral administration and for delivery of the Compounds of the invention (at least the therapeutically active moiety) either locally to the gut and/or systemically to the body providing a systemic therapeutic or biological response of the Polypeptides of the Invention, e.g. the Nanobodies, in the subject. This means that there is a sufficient amount of functional (i.e. active or not inactivated) Compound of the Invention, e.g. the Nanobodies, present in the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) to provide the desired therapeutic effect (i.e. to elicit the desired activity or the desired biological, prophylactic or therapeutic response in the subject receiving said

Compound of the Invention, e.g. the Nanobodies. The bioavailability of the Polypeptides of the Invention, e.g. the Nanobodies, in the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) and/or in the brain following administration of the composition of the invention is determined by measuring the pharmacokinetic parameters Cmax (peak concentration), AUC (area under concentration vs. time curve) and/or Tmax (time to maximal blood concentration), which are well known to those skilled in the art (Laursen et al., Eur. J. Endocrinology, 1996; 135: 309- 315). The bioavailability of the Compound of the Invention, e.g. the Nanobodies, may be determined in any conventional manner, e.g. by radioimmunoassay.

"Cmax", as used in the present invention, is the mean maximum concentration of the Compound of the invention achieved in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung), following oral administration of a single dosage of the pharmaceutical composition to the subject. Blood or bloodstream as used in the present invention, can be any form and/or fraction of blood. Without being limiting, blood or bloodstream includes plasma and/or serum. The Cmax for the Compound of the invention comprised in the pharmaceutical composition of the invention can have any value as long as said Compound of the invention provides the desired activity or therapeutic or biological response in the subject in need of said Compound of the Invention, e.g. the Nanobodies. In an aspect of the invention, the Compound of the invention reaches a Cmax in blood of at least 1 ng of Compound of the invention per ml of blood. In a further aspect, the Compound of the invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500_; 750, 100 ng or more of Compound of the Invention, e.g. the Nanobodies, per ml of blood.

In another aspect, the Compound of the Invention, reaches a Cmax in blood of at least 1 ng of Compound of the Invention, per ml of blood following oral administration of a dose of 5 mg/kg body weight of said Compound of the Invention, e.g. the Nanobodies. In a further aspect, the Compound of the Invention, reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 750, 100 ng or more of Compound of the Invention, per ml of blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.

In another aspect of the invention, following oral administration of Compound of the Invention, said polypeptide reaches a Cmax in blood of at least 1% of the Cmax that is reached following parenteral administration of the same amount of the Polypeptide of the Invention. In a further aspect, following oral administration of the Compound of the

Invention, said Compound of the invention reaches a Cmax in blood of at least 2, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50% or more of the Cmax that is reached following parenteral administration of the same amount of Polypeptide of the Invention. "Tmax". as used in the present invention, is the mean time to reach maximum concentration of the Compound of the invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) following oral administration of a single dosage of the composition of the invention. The Tmax for the

Compound of the invention comprised in the composition of the invention can have any value as long as said Compound of the invention provides the desired activity or therapeutic or biological response in the subject in need of said Polypeptide of the Invention. In an aspect of the invention, the Compound of the invention reaches the bloodstream with a Tmax of less than 120 minutes. In a further aspect, the Compound of the invention reaches the bloodstream with a Tmax of less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes. In a further aspect, the Compound of the invention reaches the brain with a Tmax of less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes.

The "concentration vs. time curve" measures the concentration of the Compound of the invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) of a subject vs. time after administration of a dosage of the composition of the invention.

In an aspect, the Compound of the invention reaches a Cmax in blood of at least 1 ng of Compound of the invention per ml of blood within less than 120 minutes following oral administration of the composition of the invention. In a further aspect, the Compound of the invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50. 100, 150, 200, 300_; 400, 500, 750, 1000 ng or more of Compound of the invention per ml of blood within less than 120 minutes following oral administration of the composition of the invention. In another aspect, the Compound of the invention reaches a Cmax in blood of at least 1 ng of Compound of the invention per ml of blood within less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of the composition of the invention. In a further aspect, the Compound of the invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of Compound of the invention per ml of blood within less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of the composition of the invention. In another aspect, the Compound of the Invention reaches a Cmax in blood of at least 1 ng of Compound of the invention per ml of blood within less than 120 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention. In a further aspect, the Compound of the invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30_; 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of the Compound of the invention per ml of blood within less than 120 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention, IB another aspect, the Compound of the invention reaches a Cmax in blood of at least 1 ng of the Compound of the invention per ml of blood within less than 90, 60, 50, 40, 30_? 20, 10, or 5 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention. In a further aspect, the Compound of the invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of Compound of the invention per ml of blood within less than 90, 60, 50, 40, 30, 20. 10, or 5 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.

The "area under the curve (AUC)", as used in the present invention, is the area under the curve in a plot of concentration of the Compound of the invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) against time. Mathematically, this value is a measure of the Integral of the instantaneous concentrations during a time interval. AUC is usually given for the time interval zero to infinity, and other time intervals are indicated (for example AUC (I₁ ^) where ti and t₂ are the starting and finishing times for the interval). Clearly blood (and/or another selected physiological compartment tissue and/or organ such as e.g. the kidney, bladder and/or lung) Compound of the invention concentrations cannot be measured 'to infinity' for a subject so mathematical approaches are used to estimate the AUC from a limited number of concentration measurements. The AUC (from zero to infinity) is used to measure the total amount of Compound of the invention absorbed by the body, irrespective of the rate of absorption. This is useful when trying to determine whether two application formulations with the same dose (for example parenteral and oral) release the same dose of Compound of the invention to the body.

The AUC for the Compound of the invention comprised in the composition of the invention can have any value as long as said Compound of the invention provides the desired activity or biological response in the subject in need of said Polypeptide of the Invention. In an aspect of the invention, the AUC for the Compound of the invention in blood following oral administration of a composition comprising said Compound of the invention is at least 500 ng/ml/minute of the Polypeptide of the Invention. In a further aspect, the AUC for the Compound of the invention in blood following oral administration of a composition comprising said Compound of the invention is at least 600, 700. 800, 900, ng/ml/minute or at least 1, 1.5, 2, 3, 4, 5. 10 or 15 μg/ml/minute of the Compound of the Invention.

In another aspect of the invention, the AUC for the Compound of the invention in blood following oral administration of a dose of 5 mg/kg body weight of said Compound of the invention is at least 500 ng/ml/minute Compound of the Invention. In a further aspect, the AUC for the Compound of the invention in blood following oral administration of a dose of 5 mg/kg body weight of said Compound of the invention is at least 600, 700, 800, 900 ng/ml/minute or 1, 1.5, 2, 3, 4, 5 or 10 μg/ml/minute Compound of the invention per ml of blood.

As discussed above, in an aspect of the invention, the bioavailability (absolute or relative) for the Compound of the invention in blood following oral administration of a composition comprising said Compound of the invention is at least 1% compared to parenteral administration of said Polypeptide of the Invention. In a further aspect, the bioavailability for the Compound of the invention in blood following oral administration of a composition comprising said Compound of the invention is at least 2, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70. 80, 90, 100% or more compared to parenteral administration of said Polypeptide of the Invention. Preferably the bioavailability (absolute or relative) for the Compound of the invention in blood following oral administration of a composition comprising said Compound of the invention is at least 5% compared to parenteral administration of said Polypeptide of the Invention.

Oral administration of one or more Compounds of the invention to a subject yields effective delivery of the Compounds of the invention to the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) to elicit the desired activity or therapeutic or biological response in the subject. In a preferred aspect of the invention, the Compound of the invention provides the prevention and/or treatment of a selected disease or condition in said subject. Accordingly, another aspect of the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the Invention, said method comprising orally administering, to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same.

In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and/or disorder.

The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a Compound of the invention to a subject suffering from said disease or disorder, said method comprising orally administering to said subject a therapeutically effective amount of the Compound of the

Invention, and/or of a composition comprising the same. Accordingly, the invention relates to the Polypeptides or compositions of the invention for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention.

in another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises oral administering, to a subject suffering from or at risk of a diseases and/or disorders that can be cured or alleviated by immunotherapy with a Compound of the Invention, a therapeutically effective amount of said Compound of the invention and/or of a composition comprising the same.

The polypeptides present in the compositions of the invention may be directed against any suitable target that is of therapeutic or diagnostic interest. The polypeptides can be functional as agonists as well as antagonists, preferably agonists. Examples include but are not limited to targets of therapeutic interest such as EPO, Growth Hormone, vWF, IL-6R. Additional examples include targets amino acid sequences that confer an increased half-life in vivo, such as a human serum protein, particularly such as human serum albumin.

The invention provides systemic delivery of the Compounds of the Invention. The desired target can be a target in any physiological compartment, tissue or organ. In an aspect the Compound of the invention is directed against a target in the kidney or the bladder and the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the invention that is directed against a target in the kidney or bladder, said method comprising orally administering, to said subject a therapeutically effective amount of said Compound of the invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Compound of the invention that is directed, against a target in the kidney or the bladder, said method comprising orally administering to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of a disease or disorder of the kidney or bladder, said method comprising orally administering to said subject a therapeutically effective amount of a Compound of the invention that is directed against a target in the kidney or the bladder and/or of a composition comprising the same. Accordingly, the invention also relates to the composition of the invention, wherein the Compound of the invention is directed against a target in the kidney or the bladder for the prevention and/or treatment of a disease or disorder of the kidney or bladder.

In another aspect, the Compound of the invention is directed against a target in the lung and the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the invention that is directed against a target in the lung, said method comprising orally administering, to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Compound of the invention that is directed against a target in the lung, said method comprising orally administering to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of a disease or disorder of the lung, said method comprising orally administering to said subject a therapeutically effective amount of a Compound of the invention that is directed against a target in the lung and/or of a composition comprising the same. Accordingly, the invention also relates to the composition of the invention, wherein the Compound of the invention is directed against a target in the lung for the prevention and/or treatment of at least one disease or disorder of the lung.

In another preferred aspect, the Compound of the invention is directed against a target on a tumor cell and the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the invention that is directed against a target on a tumor cell, said method comprising orally administering, to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Compound of the invention that is directed against a target on a tumor cell, said method comprising orally administering to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of a tumor related disease or disorder, said method comprising orally administering to said subject a therapeutically effective amount of a Compound of the invention that is directed against a target on a tumor and/or of a composition comprising the same. Accordingly, the invention also relates to the composition of the invention, wherein the Compound of the invention is directed against a target on a tumor for the prevention and/or treatment of at least one a tumor related disease or disorder. In another aspect, the Compound of the invention is directed against vWF and the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the invention that is directed against vWF, said method comprising orally administering, to said subject, a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Compound of the invention that is directed against vWF, said method comprising orally administering to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of a disease or disorder related to platelet-mediated aggregation (such as e.g. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarierectomy, carotid artery stenosis, critical limb ischaemia. cardioembolism. peripheral vascular disease, restenosis and myocardial infarction), said method comprising orally administering to said subject a therapeutically effective amount of a Compound of the invention that is directed against vWF and/or of a composition comprising the same. Accordingly, the present invention also relates to the composition of the invention, wherein the Compound of the invention is directed against vWF for the prevention and/or treatment of at least one disease or disorder related to platelet- mediated aggregation (such as e.g. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft. coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction).

In another aspect, the Compound of the invention is directed against IL-6, IL-6 receptor (IL- 6R) and/or IL-6/IL-6R complex and the invention relates to a method for the prevention and/or treatment of a subject in need of a Compound of the invention that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering, to said subject, a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Compound of the invention that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering to said subject a therapeutically effective amount of said Compound of the Invention, and/or of a composition comprising the same.

The Compounds of the invention and/or the compositions comprising the same are orally administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific Compound of the invention to be used and the pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the subject, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the oral administration of one or more Nanobodies, polypeptides or proteins of the invention, or of one or more compositions comprising the same, in one or more therapeutically effective amounts or doses. The specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.

The Nanobodies and compounds of the invention may also be used in combination with one or more further therapeutic ingredients (or pharmaceutically active compounds or principles), i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

When a second active substances or principles is to be used as part of a combined treatment regimen, it can be administered via the same oral route of administration or via a different route of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively , or according to an alternating regime). When the substances or principles are administered to be simultaneously via the same oral route of administration, they may be administered as different formulations or compositions or part of a combined formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side- effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and or a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balan.ce between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand. Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

The invention also relates to the use of a Compound of the invention for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention. The invention also relates to the use of a Compound of the invention directed against a target in the kidney or the bladder for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against a target in the kidney or the bladder. The invention also relates to the use of a Compound of the invention directed against a target in the kidney or the bladder for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the kidney or bladder. The invention also relates to the use of a Compound of the invention directed against a target in the lung for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against a target in the lung. The invention also relates to the use of a Compound of the invention directed against a target in the lung for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the lung. The invention also relates to the use of a Compound of the invention directed against a target on a tumor for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against a target on a tumor. The invention also relates to the use of a Compound of the invention directed against a target on a tumor for the preparation of a composition for the prevention and/or treatment of at least one cancer. The invention also relates to the use of a Compound of the invention directed against a target in the brain for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against a target in the brain. The invention also relates to the use of a Compound of the invention directed against a target in the brain for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the brain (such as neurogenetic diseases, (e.g. Huntington's disease and muscular dystrophy), developmental disorders (e.g. cerebral palsy), degenerative diseases of adult life (e.g. Parkinson's disease and Alzheimer's disease), metabolic diseases (e.g. Gaucher' s disease), cerebrovascular diseases (e.g. stroke and vascular dementia), trauma (e.g. spinal cord and head injury), convulsive disorders (e.g. Epilepsy) infectious diseases (e.g. AIDS dementia), obesity, diabetes, anorexia, depression, brain tumors, dementia with Lewy bodies, multi-system atrophy, progressive supranuclear palsy, frontotemporal dementia, vascular dementia or Down's syndrome). The invention also relates to the use of a Compound of the invention directed against TNF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against TNF. The invention also relates to the use of a Compound of the invention directed against TNF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease). The invention also relates to the use of a Compound of the invention directed against vWF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against vWF. The invention also relates to the use of a Compound of the invention directed against vWF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g. the formation of a non- occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary Interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral Ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction). The invention also relates to the use of a Compound of the invention directed against IL-6, IL-6R and/or IL-6/IL-6R complex for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Compound of the invention directed against IL-6, IL-6R and/or IL-6/IL-6R complex. The invention also relates to the use of a Compound of the invention directed against IL-6, IL-6R and/or IL- 6/IL-6R. complex for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM)₅ renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B- lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi^'s sarcoma, AIDS- related lymphoma, inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn^'s disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus).

As discussed above, oral administration of one or more Compounds of the invention to a subject yields effective delivery of the Compounds of the invention to the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) and/or to the brain to elicit the desired activity or biological response in the subject. In addition to the prophylactic and therapeutic response as discussed above, the Nanobodies, polypeptides and proteins of the invention may also induce other activities and biological responses. In a preferred aspect, the present invention also provides for the diagnostic use of the Polypeptides of the Invention, e.g. for in situ or in vivo labeling, such as radiolabeling and imaging. The present invention, therefore, also relates to a diagnostic method comprising the step of orally administering the Compounds of the invention and/or a composition comprising the same. In an aspect of the invention, a diagnostic method is provided comprising the steps of orally administering the Compounds of the invention and/or a composition comprising the same and in situ detecting said Polypeptides of the Invention. Detection may be done by any method known in the art.

The Compounds of the invention can be determined in situ by non-invasive methods including but not limited to SPECT and PET, or imaging methods described by Cortez-

Retamozo V, (Nanobodies: single domain antibody fragments as imaging agents and modular building blocks for therapeutics, PhD Dissertation, Vrije Universiteit Brussel, Belgium, June 2004), Arbit et al. (Eur. J. Nucl. Med. 1995; 22: 419-426.), Tamada et al. (Microbiol- Immunol. 1995; 39: 861-871), Wakabayashi et al. (Noshuyo-Byori 1995; 12: 105-110), Huang et al. (Clin. Med. J. 1996; 109: 93-96), Sandrock et al. (Nucl. Med. Commun. 1996; 17: 311-316), and Mariani el al. (Cancer 1997; 15: 2484-2489). These in vivo Imaging methods may allow the localization and possibly quantification a certain target, for example, by use of a labeled Compound of the Invention, specifically recognizing said target. In vivo multiphoton microscopy (Bacskai et al.. J. Cereb. Blood Flow Metab. 2001; 22: 1035-1041) can be used to image the presence of a certain target with labeled Compounds of the invention specific for the target.

The Compound of the invention orally administered in the diagnostic methods of the invention may be labeled by an appropriate label. The particular label or detectable group used in the method is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the Compound of the invention used in the method. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and, in general, almost any label useful in such methods can be applied to the method of the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means. Useful labels in the present invention include but are not limited to magnetic beads (e.g. Dynabeads^IM), fluorescent dyes (e.g. fluorescein isolhiocyanate, Texas red, rhodamine, Cy3, Cy5, Cy5.5, Aϊexi 647 and derivatives), radiolabels (e.g.³H,¹²⁵1,³⁵S,¹⁴C,³²P or "¹¹¹Tc), enzymes (e.g. horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the Compound of the invention according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, the available instrumentation and disposal provisions. Non-radioactive labels are often attached by indirect means. Means for detecting labels are well known in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of a photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photo multipliers and the like.

Finally, although the use of the Compounds of the invention (as defined herein, e.g. the

Nanobodies and/or constructs comprising said Nanobodies) is much preferred, it will be clear that on the basis of the description herein, the skilled person will also understand that other (single) domain antibodies, as well as polypeptides and proteins comprising such (single) domain antibodies (in which the terms "domain antibody", "single domain antibody" and "dAb" have their usual meaning in the art) are also encompassed within the scope of the present invention.

The invention will now be further described by means of the following non-limiting experimental part.

EXPERIMENTAL PART

Examfije 1» - Identification of Nanobodies binding to human FeRn General The major histocompatibility complex class I-related receptor FcRn was first identified as the receptor that transports maternal IgGs from mother to young via the neonatal intestine. However recent data have indicated that the neonatal receptor is also responsible for rescuing IgG and albumin from degradation and therefore prolong their half-lives (Andersen et ah. 2006; Anderson et al, 2006; Ghetie and Ward, 2000; Kim et a!.. 2006; Lencer and Blumberg, 2005; Ober et al., 2004a; Ober et al., 2004b). FcRn is expressed inside endothelial cells that line blood vessels, mainly in early/recycling endosomes, where IgG and albumin can be internalized by fluid phase endocytosis. To a minor extent FcRn is also expressed in the cell surface. IgG and albumin bind independently to FcRn in a pH-dependent manner, with binding at pH 6.0 but not at pH 7.4. The acidic environment of the endosomes facilitates the interaction. Bound IgG and albumin are recycled back to the surface and released from the cell, while unbound ligands are shuttled downstream to lysosomal degradation (Ghetie and Ward, 2000).

The role of FcRn as an IgG transporter opens the opportunity to generate new therapeutics for modulation of IgG levels, as it is desired in the case of autoimmune diseases. Because the transport and protection of IgG are dependent on its Fc-domain, it can be proposed that small molecules or peptides with therapeutic activities could be fused to Fc fragments and therefore delivered across the epithelium and have long circulating long lives. Moreover, the fact that FcRn is expressed on many epithelial surfaces in adult humans including the lungs (Spiekermarm et al., 2002: Bitonti and Dumont, 2006), suggests that FcRn transport pathway could be used as a delivery system of therapeutic agents by non-invasive means (i.e. aerosols administered into the lungs using normal breathing maneuvers).

Structure FcRn comprises a heterodimer of beta2-microglobulin and a 45 to 53 KDa protein. All three extracellular and membrane domains of FcRn share homology with the corresponding regions of major histocompatibility complex (MHC) class I molecules, with much less homology between the cytoplasmic domains. The X-ray crystallographic structure of the extracellular domains of FcRn confirmed that it is structurally similar to MHC class I molecules (Ghetie and Ward, 2000).

The FcRn-IgG interaction depends on conserved histidine residues in the IgG-Fc part that interact with negatively charged residues in the beta-2 domain of the hFcRn heavy chain. Recent studies showed that conserved H 166 in the hFcRn heavy chain, directly opposite to the IgG binding site, is a key player in the FcRn- albumin interaction (Andersen et al, 2006).

Disease relation The fact that FcRn regulates IgG homeostasis, modulation of FcRn function and/or expression might be an effective approach for the treatment of autoimmune diseases.

It has been suggested that deregulation of FcRn expression may be involved in situations in which hypercatabolisni is observed, such as after burns and in myotonic dystrophy. It is also possible that some types of IgG deficiencies such as familial idiopathic hypercatabolism may be caused by abnormalities in FcRn expression or function (Ghetie and Ward, 2000).

Identification of nanobodies binding bFcRn:

Members:

Families of binders: members: -I 215~C3, 215-E2, 215-B5 -II 215-B3 -III 216-E11 -IV 215-A4 -V 218-C5, 218-A5, 216-E6, 218-H4 -VI 265-E6, 265-E4, 265-F3

Families 1 to IV are binding hFcRn HC while families V and VI are binding also shFcRn.

Construct: Bivalent FcRn nanobody: 218-H4 20GS 218-H4 Sequences:

Example 1.1: Animal Immunizations hFcRn HC and shPcRn mutmix were kindly provided by Inger Sandlie (University of Oslo, Norway). Llama 153 was immunized, according to standard protocols, with 6 boosts of a cocktail 1 12 containing hFcRn HC (only the human FcRn heavy chain). Llama 154 was immunized, according to standard protocols, with 3 boosts of a cocktail 116 containing shFcRn mutmix (intact soluble FcRn, heavy chain and beta2- microglobulin). For animal 153, blood was collected 4 and 7 days after boost 6. In addition, approximately 1 g of lymph node was collected from this animal 4 days after boost 6. For animal 154, blood was collected 22 days after boost 3.

Example 1.2: Library construction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 1.3: Selections of phage displaying faFeRn binding Nanobodies Phage library 153 was used for selection at pH 5 on hFcRn heavy chain (hFcRn HC) while phage library 154 was used for selection at pH 5 on shFcRn (heavy chain and beta2- microglobulin). Both hFcRn proteins were immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control) in PBS at pH 7.4. After 2 hours blocking with 4% Marvel PBS the plates were washed several times with PCA buffer/Tween pH 5.1 (1OmM Sodium citrate+10 niM SodiuiB phosphate+10 mM Sodium acetate +115 mM NaCl/ Tween pH 5.1). To minimize the number of phage binding to the albumin binding site of the FcRn protein the phage was pre-incubated with 250 ug/ml human serum albumin in 2 % Marvel PCA buffer pH 5.1. Following incubation with the phage libraries and extensive washing with pH 5.1 buffer, bound phage was eluted with trypsin. The eluted phage were amplified and applied in a second round of pH 5 selection on 5 ug/ml. 0.5 ug/ml and 0 ug/ml (control) immobilized hFcRn proteins. To minimize the number of phage binding to the albumin binding site of the FcRn protein the phage was pre-incubated with 250 ug/ml human serum albumin in 2 % Marvel PCA buffer pH 5.1. Individual colonies obtained from the eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (peripiasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein). Selections on shFcRn using libraries 153 and 154 were also performed at neutral pH.

shFcRn (C48S/C251S) was immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 10 ug/ml, 1 ug/ml and 0 ug/ml (control) in PBS at pH 7.4. After 1 hour blocking with 4% Marvel PBS the plates were washed several times with PBS /Tween. To minimize the number of phage binding to b2-microglobulin the phage was pre-incubated with 12-100 ug/ml b2-micro globulin from human urine (SIGMA M4890, 126kl 151). Following incubation with the phage libraries and extensive washing with PBS/Tween and PBS, bound phage were eluted with trypsin. The eluted phage were amplified and applied in an identical second round selection on 10 ug/ml, 1 ug/ml and 0 ug/ml (control) immobilized shFcRn protein. Individual colonies obtained from the eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (peripiasmic extracts) according to standard methods.

Example 1.4: Screening for faFeJRn HC binding Nanobodies In order to determine binding specificity to hFcRn HC, the clones were tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to hFcRn HC was tested. Shortly, 0.5 ug/ml hFcRn HC was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. After washing with PCA ph 5.1 buffer, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ui 2% Marvel PCA pH 5.1 were allowed to bind to the immobilized antigen. After incubation and several wash steps with pH 5.1 buffer,, phage binding was revealed using a HRP-conjugated monoclonal-anti-M 13 antibody (Gentaur Cat# 27942101) in 1 % Marvel PCA pH 5.1. The same ELISA assay was performed at neutral pH by using PCA pH 7.4 buffer. Binding specificity was determined based on OD values compared to controls wells having received an irrelevant phage or no phage. Figure 1 shows a selection of clones binding to hFcRn HC at pH 5.1 and pH 7.4.

Example 1.5: Screenirag/ϊdeEtificatioϊi of sfaFcRn biiidmg_Njnobodies

In order to determine binding specificity to shFcRn, the clones were tested in an ELlSA binding assay setup, using the monoclonal phage pools obtained from the pH 5 and neutral pH selections.

Phage binding to shFcRn was tested. Shortly, 0.5 ug/ml shFcRn was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. After washing with PCA pH 5.1 buffer or PBST, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PCA pH 5.1 or PBST were allowed to bind to the immobilized antigen. After incubation and several wash steps with pH 5.1 or PBST buffer, phage binding was revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat# 27942101) in 1 % Marvel PCA pH 5.1 or PBS. A binding ELISA against b2-microglobulin from human urine (SIGMA M4890. 126kl 151) was also performed in parallel.

Binding specificity was determined based on OD values compared to controls wells having received an irrelevant phage or no phage.

Figure 2 shows a selection of clones binding to hFcRn HC and shFcRn at pH 5,1 (left bar). Figure 3 shows a selection of clones binding to shFcRn at neutral pH.

The strategy of the generation of bi- or multivalent Nanobodies has previously been successfully applied to target other proteins and may result in a significant increase in avidity (Zhang et al, 2004; Coppieters et al., 2006; Roover et al., 2007). Thus, a bivalent FcRn nanobody was constructed using a PCR-based method where two copies of the same nanobody (218-H4, that previously showed binding to FcRn by SPR analysis) were connected by a 20 Gly-Ser linker (four repetitive GGGGS) -see SEQ ID NO: 112, clone 218-H4_20GS_218-H4.

Example 1.6: Determination of transcytotie efficacy of Natsobody in cell models Transcytosis assay using A375 cells stably transfected with shFcRn. Such characterized cells are used to study whether the isolated Nanobodies bind in a pH dependent manner to membrane anchored receptors, and how the ligands, IgG and albumin, affect the binding.

This assay is preformed as follows:

12mm Transwell filters (0.4 urn pore size, Costar/Corning, Acton, MA, USA) are inoculated with 5* 10^s cells. Cells are grown overnight to confluence, washed with PBS and placed in a new 12- well plate with 1.5 ml medium in the basolateral and 0.5 ml in the apical compartment. The medium in the loading compartment contains anti-FcRn nanobodies (monovalent, multivalent, e.g. bivalent, multispecific) to be transported and 125 pg/m] streptavidin-HRP (Sanquin) to assess non-receptor-mediated transport or leakage. Samples of 1 OOul are taken from the appropriate compartment, and nanobodies are quantified. Apical to basolateral transport is calculated according to ([Nanobodiesjbasolateral* 1 .5ml)/(nanobodies]input* 0.5ml) * 100%, and basolateral to apical transport as

([nanobodiesjapical* 0.5ml)/([nanobodies]input* 1.5ml) * 100%. IgGs can be used as a control for transcytosis. As a positive control IgGs are used and as a negative control irrelevant nanobodies are used. In addition, experiments are performed in which IgGs will be co-incubated with anti-FcRn nanobodies (Trojan horse concept).

Example 1.7: Determination of shFeRn binding properties of seleeted Nanobβdy candidates by ELISA

Wells were coated with 100 μl of anti-myc (monoclonal, mlgGl) at 1.0 μg/ml, and incubated overnight at 4°C. They were then blocked with 4% skimmed milk (Acumedia) for 1 h and washed four times with PBS/0.005% Tween 20 (PBS/T) pH 7.4. Nanobody candidates were diluted to 2 μg/ml in 4% skimmed milk/PBS/T and added to the wells. After incubation for 1.5 h at room temperature the wells were washed four times with PBS/0.005%/T ween 20 (PBS/T) pH 7.4. GST-fused shFcRn (0.5 μg/ml; eukaryoticly expressed; JTA) pre-incubated with an HRP conjugated polyclonal anti-GST from goat (1 :5000; GE Healthcare) in 4% skimmed milk PBS/0.005% Tween 20 (PBS/T) pH 7.4 was added to each well and incubated for 1.5 h at room temperature followed by four times washing with PBS/0.005%/Tween 20 (PBS/T) pH 7.4. 100 μl of the substrate ABTS/H₂O₂ (Sigma) was added to each well. The absorbance was measured at 405 run using a Sunrise TECAN spectrophotometer (TECAN. Maennedorf, Switzerland). The same ELISA was repeated with PBS/T pH6.0. The same ELISA was repeated with PBS/T pH6.0 with serial dilutions of the Nanobody candidates ranging from 0.016-4.0 μg/ml. Soluble human β2-microglobulin (Abeam) was coaled at 5 μg/ml coated in each well and incubated overnight at 4°C. Wells were then blocked in with 4% skimmed milk (Acumedia) for I h and washed four times with PBS/0.005% Tween 20 (PBS/T) pH 6.0. Nanobody candidates were diluted to 2 μg/ml and pre-incubated with an HRP conjugated anti rnyc antibody (monoclonal, mlgGl, Serotec) in 4% skimmed milk/PBS/T and added to the wells. After incubation for 1.5 h at room temperature the wells were washed four times with PBS/0.005%/Tween 20 (PBS/T) pH 6.0. 100 μl of the substrate TMB (Calbiochem) was added to each well. The absorbance was measured at 620 ran using a Sunrise TECAN spectrophotometer (TECAN. Maennedorf, Switzerland). An polyclonal anti human β2-microglobulin antibody preparation (diluted 1 :2000; Sigma) and an anti human 02- microglobulin monoclonal antibody (1 :2000; Sigma) were included as positive controls. They were visualized by HRP conjugated protein G (Calbiochem) (see Figure 4). Furthermore, In order to determine whether the bivalent nanobody 218-H4_20GS_218-H4 was binding to hFcRn and to compare this binding with the correspondent monovalent nanobody the following ELISA was performed:

1 ug/ml shFcRn was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked for 2 hours using 4% Marvel skimmed milk in PBS. After washing with PCA buffer pH 5.1 monovalent and bivalent purified naπobodies (0-50 nM in 2% Marvel/PCA pH 5.1) were added to immobilized hFcRn, After one hour incubation the plate was washed several times with the low pH buffer and 1 ug/ml of anti-myc (in 1% Marvel/PCA pH 5.1) was added for another hour. After some washing bound monovalent and bivalent nanobodies were revealed using an anti mouse-HRP antibody (Dako, P 0260). A similar binding ELISA against b2~microglobuh^'n from human urine (SIGMA M4890, 126kl 351) was also performed in parallel.

Figure 22 shows the results of the binding to shFcRn and b2-microglobulin ELISAs at pH 5.1. Bivalent 218-H4 nanobody showed up to a 10 fold improvement in binding to shFcRn when compared to the monovalent nanobody. None of the nanobodies (mono or bivalent) were binding the b2-microglobulin indicating that the nanobodies are binding the FcRn heavy chain.

Example_1.8: Determination of shFcRn binding properties of selected Nanobody candidates by SPR

SPR experiments were carried out using a Biacore 3000 instrument (GE Healthcare). Flow cells of CM5 sensor chips were coupled with shFcRn-GST (~ 800 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 10 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5 or pH 7.4, or HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA. 0.005% surfactant P20) at pH 7.4 (Biacore AB) were used as running buffer and dilution buffer. For binding to immobilized shFcRn-GST, 200 nM of each Nanobody was injected over the surface at constant flow rate (10 μl/ml) at 25 °C . In all experiments, data were zero adjusted and the reference cell subtracted (see Figure 5).

4/10 selected Nanobody candidates bind Io shFcRn-GST under both pH conditions, pH 7.4 was well as pH 6.0, and they bind to heterodimeric GST-fused shFcRn and not to soluble human β2-microglobulin at pH 6.0. Thus, it is likely that the FcRn positive candidates interact with the folded human FcRn heavy chain.

The SPR analyses (Figure 5) are in agreement with the obtained in ELISA (Figure 4). The differences in RU responses between pH 6.0 and pH 7.4 may indicate that the interactions of the selected Nanobody candidates bind slightly stronger at acidic pH than at physiological pH.

Selected Nanobody candidates that bind to shFcRn: - 218-A5 - 218-H4 - 216-E6 - 218-C5 Selected Nanobody candidates that do not bind to shFcRn: - 215-C3 - 215-E2 - 215-B5 - 215-B3 - 216-E1 1 - 215-A4

Irrelevant Nb (Nb=nanobody) In a further experiment, SPR experiments were carried out using a Biacore 3000 instrument (GE Healthcare). Flow cells of CM5 sensor chips were coupled with shFcRii-GST

(~ 800 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 10 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5 or pH 7.4, or HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) at pH 7.4 (Biacore AB) were used as running buffer and dilution buffer. For binding to immobilized shFcRn- GST, 200 nM of each Nanobody was injected over the surface at constant flow rate (10 μl/ml) at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted. Data evaluation was performed using BIAevaluation 4.1 software (BIAcore AB).

Result (Figure 23): 4/8 selected Nb candidates bind to shFcRn-GST under both pH conditions. pH 7.4 as well as pH 6.0.

The differences in RU responses for some of the Nb candidates pH 6.0 and pH 7.4 may indicate that they bind slightly stronger at acidic pH than at physiological pH.

Selected Nanobody candidates that bind to shFcRn:

- 265-F3 - 218-Gl - 218-Cl - 2162-E11

Selected Nanobody candidates that do not bind to shFcRn: - 215-E2 - 265-C4 - 265-E6 - 265-E4

All selected Nanobody candidates were then injected at 200 iiM over the same surface with immobilized shFcRn at pH 6.0 or pH 7.4, The binding responses obtained are summarized in Table B-2.

. b: Binding of the Nbs to immobilized shFcRn at pH 7.4. c; Binding of the Nbs to immobilized shFcRn at pH 6.0. d: FcRn binding activity: +^■++ indicates high activity, ++ indicates intermediate activity, + indicates low activity and - indicates no activity. Evaluation of FCRB binding properties of monomelic and dϊmerie Nb218-H4 variants

Two strategies were explored:

1. Direct binding of the monomeric and dimeric versions of Nb 218-H4 to immobilized shFcRn at pH 6.0 as well as pH 7.4.

2. Indirect binding of shFcRn-Nb 218-H4 complexes to immobilized human IgG at pH6.0.

Strategy 1 and 2 are schematically illustrated in Figure 24A and 24B, respectively.

Strategy 1:

Material and methods:

SPR experiments were carried out using a Biacore 3000 instrument (GE healthcare). Flow cells of CM5 sensor chips were coupled with shFcRn-GST (~ 1000 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 2 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 was used as running buffer and dilution buffer. Monomeric 218- H4 (100 nM), dimeric 218-H4-20GS-218-H4 (25-50 nM), monomeric and dimeric Nb with irrelevant specificity (directed against the mouse Epo receptor; 25-50 nM) were injected at a flow rate of 50 μl/ml at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted. Data evaluation was performed using BIAevaluation 4.1 software (BIAcore AB).

The SPR assay was repeated with HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl₅ 3 mM EDTA, 0.005% surfactant P20) pH 7.4 (Biacore AB).

Results: Figure 25

Strategy 2:

Material and methods: SPR experiments were carried out using a Biacore 3000 instrument (GE healthcare). Flow cells of CM5 sensor chips were coupled with anti-NIP human IgGl (~ 1200 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 5 μg/ml of the protein in 10 niM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 niM phosphate buffer. 0.15 M NaCl. 0.005% Tween 20) at pH 6.0 was used as running buffer and dilution buffer. Monomeric 218- H4 (500 nM), dimeric 218-H4-20GS-218-H4 (500 nM), monomeric and dimeric Nb with irrelevant specificity (directed against the mouse Epo receptor (261-H3-20GS-261-H3); 500 nM) were pre-incubated with 100 nM monomeric shFcRn (Andersen et al.. FEBS, 2008) before injection at a flow rate of 50 μl/ml at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted. Data evaluation was performed using BIAevaluation 4.1 software (BIAcore AB).

Results: Figure 26

Conclusions:

Both SPR strategies show that the bivalent Nb 218-H4, i.e. polypeptide with SEQ IB NO: 112. is fully functional. It binds bivalent) y at both pH 6.0 and pH 7.4 (strategy 1), while no binding was detected for a bivalent Nb with irrelevant specificity at either pH.

The bivalent molecule does not interfere with binding of shFcRn to immobilized human IgGl at pH 6.0 (Strategy 2). No (monomeric or bivalent) binding was observed for the dimeric irrelevant Nb control.

w Kinetic determination of the Nb 218-H4 candidate, Le. polypeptide with SEQ ID NO:37

Material and methods:

SPR experiments were carried out using a Biacore 3000 instrument (GE healthcare). Flow cells of CMS sensor chips were coupled with shFcRn-GST (~ 1000 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 2 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 rnM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 was used as running buffer and dilution buffer. Serial dilutions of monomeric 218-H4 (500 nM) was injected (500-15 nM) at a flow rate of 50 μi/ml at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted. Data evaluation was performed using BI Ae valuation 4.1 software (BIAcore AB).

The SPR kinetic assay was repeated with HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) pH 7.4 (Biacore AB).

Results: The data obtained in Figure 27 were used to calculate the affinity of Nb 218-H4 towards shFcRn at pH 6.0 and pH 7.4 using the BIAevaluation 4.1 software. Two models were explored: the simple Langmuir 1:1 model and the state-state affinity model.

Conclusions:

The sensorgram obtained for binding of Nb 218-H4 to immobilized shFcRn fitted very well to a simple 1:1 Langmuir model. The binding affinity was derived to be approx. 52.5 nM and 21.2 nM at pH 7.4 and pH 6.0, respectively. Thus, the binding affinity is 2 fold stronger at pH 6.0 than at pH 7.4. This can be explained by the small difference in the kinetic constants shown on table X. Using the equilibrium binding responses in a state-state affinity model gave rise to 55.6 nM and 22 nM for binding to shFcRn at pH 7.4 and pH 6.0, respectively. Thus, the two kinetic binding models used, gave approx. the same kinetic binding values.

Example 1.9: Influence of of the presence of IgG and HSA on binding of Nanobody candidates of human FcRn

To evaluate whether binding of selected Nanobody candidates to shFcRn interfere or discriminate with pH dependent binding to IgG and HSA. SPR experiments were carried out using a Biacore 3000 instrument (GE healthcare). Flow cells of CM5 sensor chips were coupled with MgGl (~ 1000 RU) or monomeric HSA (~ 1000 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 10 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 was used as running buffer and dilution buffer. For competitive/additive binding analyses, monomeric shFcRn (prokaryoticiy expressed; JTA) was pre-incubated with Nanobody candidates and injected over immobilized hlgGl or HSA at a flow rate of 10 μl/ml at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted (see Figure 6).

FcRn binds both IgG and HSA in the presence of the four positive anti-FcRn binding Nanobody candidates (Nb218-A5, Nb218-H4, Nb216-Eό and 218-C5). This is demonstrated in the SPR assay as additive resonance binding responses (RU). Thus, the Nanobodies selected towards shFcRn do not interfere with ligand binding (IgG and HSA) to shFcRn at acidic pH.

m Influence of the presence of IgG and HSA OB binding of Nb 218-H4 to human FcRn at pH 6.0 aed pH 7.4

Material rad methods:

SPR experiments were carried out using a Biacore 3000 instrument (GE healthcare). Flow cells of CM5 sensor chips were coupled with shFcRn-GST (~ 1000 RU) using amine coupling chemistry as described in the protocol provided by the manufacturer. The coupling was performed by injecting 2 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare). For all experiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 was used as running buffer and dilution buffer. 300 nM monomeric Nb 218-H4, 100 nM anti-NIP human IgGl or 10 μM HSA was injected alone or 100 nM Nb 218-H4 was pre-incubated with 100 nM human IgG or 10 μM HSA before injected at a flow rate of 50 μl/ml at 25⁰C. In all experiments, data were zero adjusted and the reference cell subtracted. Data evaluation was performed using BIAevaluation 4.1 software (BIAcore AB). The SPR kinetic assay was repeated with HBS-EP buffer (0.01 M HEPES, 0.15 M NaCI, 3 mM EDTA₅ 0.005% surfactant P20) pH 7.4 (Biacore AB).

Results (Figure 28): All combinations generated additive binding responses. Thus, the data indicate that Nb 218-H4 does not interfere with ligand (human IgG and HSA) binding to shFcRn at pH 6.0 or pH 7.4. The FcRn ϊigands, i.e. human IgG and HSA, do not bind to ShFcRn at pH 7.4 while Nb 218-Nb does.

Example 1.10: Nanobody candidates as FeRa detection reagents: influence on ligand binding.

In Example 1.9 it was shown that the FcRn binding Nanobodies bind independently of the presence of IgG and HSA. These results may suggest that the selected Nanobodies can be used as FcRn detection molecules in in vitro assays. The Nanobody candidates also bind shFcRn at both pH 6.0 and pH 7.4.

The following experiment is done to evaluate whether selected Nanobody candidates can be used as detection reagents in FcRn-ligand binding assays.

Wells were coated with lOO μl of BSA-5~iodo-4-hydroxy»3-nitro-phenacetyl (NlP) conjugate (NIPs₆BSA) at 1 μg/ml, and incubated overnight at 4⁰C. They were then blocked with 4 % skimmed milk for 1 h and washed four times with PBS/0.005% Tween 20 (PBS/T). Anti-NIP hlgGl was diluted in 4 % skimmed milk/PBS/T and added in concentrations ranging from 0.004-1 μg/ml, incubated for 1 h at room temperature and washed four times with PBS/T at pH 6.0. GST-fused shFcRn (0.5 μg/ml) was diluted in 4 % skimmed milk/PBS/T at pH 6.0 and added to the wells. After incubation for 1 h at room temperature the wells were washed four times with PBS/T at pH 6.0. Nanobody candidates (1 μg/ml) pre-incubated with an HRP conjugated anti-myc antibody (1 : 1000, mlgGl, Serotec) were diluted in 4 % skimmed milk/PBS/T at pH 6.0 and added and then incubated for 1 h at room temperature. Bound Nanobodies were visualized by TMB substrate (Calbiochem). The absorbance was measured at 450 nm using a Sunrise TECAN spectrophotometer (TECAN, Maennedorf, Switzerland). The ELISA wet up was also performed with HSA (Sigma, HLPC purified) coated in concentrations ranging from 0.2-50 μg/ml. The FcRn binding Nanobodies can be used as FCRB detection reagents in ligand binding ELISA assays.

4 out of 10 tested Nanobody candidates bind shFcRii. Nb218-A5, Nb218-H4, Nb216-E6 and Nb218-C5 bind shFcRn and not human β2-microglobulin. Thus, it is likely that these anli- FcRn candidates bind to the human FcRn heavy chain.

The Nanobodies bind shFcRn independently of shFcRn binding to IgG and HSA. This means that the binding sites for the selected Nanobodies are distal from the IgG and HSA binding sites on the heavy chain α2-domain.

There is a great need for FcRn detection reagents, few antibodies exist commercially. The 4 anti-FcRn binding Nanobodies can be used as detection reagents for FcRn at both pH 7.4 and pH 6.0, and be used as detection reagents in ligand binding analyses. The anti-FcRn Nanobodies may be tested in flow cytometry for determination of FcRn expression profiles (we have both FcRn positive and negative cells). Such FcRn detection reagents can be used for several applications. Cross-species binding to a soluble mouse form of FcRn can be performed. Kinetics of can be performed.

Example 1.11: Nartobody binding hFcRn and their characterization in traitscytosis and recycliϊig/regciie

For anti-hFcRn nanobody (hFcRn nanobody) transport experiments, 12mm Transwell filters (0.4 μm pore size, Costar/Corning, Acton, MA, USA) are inoculated with 5* 10^Λ5 cells. Cells are grown overnight to confluence, washed with PBS and placed in a new 12- well plate with 1.5 ml medium in the basolateral and 0.5 ml in the apical compartment. The medium in the loading compartment contains anti-hFcRn nanobody to be transported and 125 pg/ml streptavidin-HRP (Sanquin) to assess non-receptor-mediated transport or leakage. Samples of lOOμl are taken from the appropriate compartment after two hours, and IgG is quantified by ELISA. Apical to basolateral transport is calculated according to ([hFcRn nanobody]basolateral* 1.5ml)/([ hFcRn nanobody] input* 0.5ml) * 100%, and basolateral to apical transport as ([hFcRn nanobody] apical* 0.5ml)/([ hFcRn nanobody] input* 1.5ml) * 100%. For hFcRn nanobody recycling the assay is performed for 24 hours, and hFcRn nanobody is quantified in both compartments. All experiments are performed in triplicate. hFcRn nanobody quantification hFcRn nanobody concentrations are determined by sandwich ELISA,

Example 1.12s In vivo models foojrgl_to_svstemic or pulmonary to systemic Mice model

A valuable model to study the oral to systemic or pulmonary to systemic delivery of FcRn- specific nanobodies are the human FcRn transgenic mice generated as described in Roopenian et al., The Journal of Immunology, 2003, 170: 3528-3533. Such mice have been used by others to study the systemic delivery of human antibodies or derivatives adminstered topologically (Spiekermann et al., J. Exp. Med.. 2002, 196: 303-310).

Nanobodies thai can e.g. be tested

Anti-mouse or human EpoReceptor nanobodies (see below) Anti- mouse or human EpoReceptor nanobodies linked to anti-hFcRn nanobodies

Epo linked to anti- mouse or human FcRn nanobodies.

Anti-mouse Leptin Receptor with/ without half-life extension optionally linked to anti- mouse FCRΏ nanobodies,

Methodology

Mice are anaesthetized with Isoflurane by inhalation and Epo fusion or nanobody proteins are fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart as indicated), or administered intranasally by instilling a total volume of 14 microliter into the nostrils. Intragastric (i.g.) proteins were administered with 80-400 ug of soybean trypsin inhibitor in 100-500 ul carbonate buffer, pH 8.8, for mice 10-d or 4-wks-old, respectively. Proteins administered by nose were suspended in PBS alone. Mice were killed by CO2 inhalation 8 h or 4 d later and whole blood was obtained by cardiac puncture.

To examine the in vivo effect of the Epo or the nanobody proteins, whole blood samples were added to ReticOne reagent according to the manufacturer's instructions. Flow cytometry was performed with a Coulter Epics XL machine. Acquisition parameters were calibrated each time by Retic-Cal Biological Calibration and Retic-C Cell Control. 40,000 total events in the red blood cell gate were acquired and analyzed with ReticOne automated software for percentage of reticulocytes (all materials from Beckman Coulter). Oral administartion in other models and human volunteers

A similar approach for oral administratrion is also possible in large animals like monkeys and also humans. Animals or human volunteers adhere to a clear liquid diet from eight hours until four hours prior to oral administration at which time they consume a standard meal. They then fast for four hours after which the Epo fusion or nanobody proteins is consumed, reconstituted in 250 ml of an isosmotic polyethylene glycol solution containing 421 mg of sodium bicarbonate. The polyethylene glycol is used as a nonabsorbable transit marker (Leyerly et al, Infection and Immunity, 1991, 89: 2215-2218: Kelly et al., Antimicrobial Agents and Chemotherapy, 1997, 41 : 236-241 ; Warny et al., Gut, 1999, 44: 212-217). Enteric capsules could also be used.

Pulmonary administration in non —human primates and human volunteers

Animals are anesthetized with a combination of Valium and ketamine, and aerosols are administered directly into the lungs through an endotracheal tube. An Aeroneb Pro nebulizer (AeroGen, Mountain View. CA) (mass median aerodynamic diameter of 4.1 urn and geometric standard deviation of 3.1 um, as measured with an Andersen Cascade Impactor, ThermoAndersen, Smyrna, GA) is used in-line with a Bird Mark (Bird Products,, Palm Springs, CA) 7A respirator, to administer aerosols to monkeys. Aerosols are generated until ~2 ml of the drug solution per animal had exited the nebulizer (~5 min). For shallow breathing, animals are regulated at 25-32 breaths per min (pressuresetting of 12-15 cm_H20 on the respirator) and for deep breathing, 20 breaths per min with a 3-sec breath hold (pressure setting _ 25-28 cm H2O). Additionally, a nebulizer that produces smaller aerosol particles (Bird Micronebulizer, mass median aerodynamic diameter of 2.5 um) is used for deep-breathing animals, to direct deposition deeper into the lung (Bitonti et al., PNAS, 2004, 101 : 9763-9768).

Pulmonary administration in human volunteers can be achieved with an Aeroneb® Pro (Aerogen, Inc., Mountain View. CA) nebulizer that generated an aerosol, from a solution of proteins in PBS, with a mass median aerodynamic diameter (MMAD) of 4.2 um and a geometric standard deviation (GSD) of 3.1 um. Volunteers are requested to breathe at 10- 30% of vital capacity (VC) and a rate of approximately 15 breaths per minute (BPM) to favor deposition of the protein to the central airways. Volunteers are first trained on breathing maneuvers Io achieve the target parameters, then the protein solution is loaded into the nebulize! cup and dosing is performed. Inhalation is monitored using a pressure sensing system thai converted pressure readings to flow rate data.

Example 1.12: FcRn model to studv half-live in vivo

The elimination of anti-FcRn nanobodies in vivo can be tested using two different mouse transgenic lines: FcRn^{" "} hFcRn (276) Tg (cDN A transgenic line 276) and FcRn^{" "} hFcRn (32) Tg (genomictransgenic line 32), Mice caring one (hFcRn Tg) or two copies (BFcRn Tg/Tg) of the hFcRn Tg were included in the study. Both transgenic and control mice (FcRn^{" "} and FcRn^{+ "}) are injected on day 0 with a single i.p. injection of Nanobodies. Blood samples are obtained from the retroorbital plexus using non-hep arinized capillary pipettes 5 min before injection and several time points after the injection. An ELISA is used to monitor the serum concentrations of the nanobodies (Petkova et al., 2006, International Immunology, 18: 1759-1769).

A surrogate model to study half-life

The A375 melanoma cell can form tumors in immune deficient mice like nu/nu, SCID and NOD/scid. It seems reasonable that in such a mouse that carries a large A375 tumor that expresses human FcRn, human antibodies or anti-FcRn nanobodies will become concentrated at the side of the tumor. Because of the natural role of FcRn in extending the half-life it might be expected that in such tumor carrying mice the half life of the nanobodies will increase.

Mice with an A375 and A375-hFcRn (see transcylosis assay) tumor of sufficient size are injected with an anti-hFcRn nanobody on day 0 with single i.p. injection of Nanobodies. Blood samples are obtained from the retroorbital plexus using nαn-heparinized capillary pipettes 5 min before injection and several time points after the injection. An ELISA is used to monitor the serum concentrations of the nanobodies.

Review articles on FCRn Anderson CL, Chaudhury C, Kim J Bronson CL, Wani MA and Mohanty S.

Perspective-FcRn transport albumin: relevance to immunology and medicine. TRENDS in Immunology (2006). 27: 343-348 Ghetie V and Ward SE. Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu. Rev. Immunol. (2000) 18: 739-766 Lencer WI and Blumberg RS.

A passionate kiss, then run: exocytosis and recycling of IgG by FcRn. TRENDS in cell biology (2005). 15: 5-9.

Example 2: - ϊdeatifieation of pIgR binding Naitobodies/phages and IgA competitors.

Sequences:

Example 2.1: Immsinkatlons

Two llamas (097 and 098) were immunized with 6 boosts of R&D Systems Cat # 2717-PG, which is the ectodomain of human plgR, according to standard protocols. Blood was collected from these animals after 7 days after boost 6 and 10 days after boost 6.

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4⁰C for further use.

Exam£|e_23^Se^tiMis Phage libraries from llama's 097 and 098 were used for selections for two rounds on ectodomain of plgR (R&D Systems Cat # 2717-PG). plgR was immobilized directly on Nunc Maxisorp ELISA plates at 5 microg/ml or 1 ug/ml and 0 ug/ml (low control) for the first round of selection and 5 microg/ml or 0.5 ug/ml and 0 ug/ml (low control) for the second round of selection. Binding phages were retrieved from both first and second selection rounds using trypsin elution, IgA specific elution and BSA specific elution (neg. control).

Specific elution was performed by incubating the wells with 150 ug/ml IgA for 1 hour, thereby replacing Nanobodies binding on the IgA binding spot of plgR.

For the second round of selection phages from the output of the first round of selection eluted with IgA were used.

Output of both Rl and R2 selections were analyzed for enrichment factor (# phage present in eluate relative to control). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ~ 80 ul) were prepared according to standard methods (see for example the prior art published and applications filed by Abiynx N.V.). Example 2.4: screening

In order to determine binding specificity to plgR, the clones were tested in an ELISA binding assay setup. In short, 5 ug/ml plgR ectodomain was immobilized on Maxisorp microliter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 ul of periplasmic extract containing Nanobody of the different clones in 100 ul 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). Overall more than 70% of the selected clones were able to bind to plgR with some specificity (signal more than 2x above background).

Example 2.5: screening for competition In order to determine IgA competition efficiency of plgR binding Nanobodies the positive clones of the binding assay were tested in an ELISA competition assay setup.

In short. 5 ug/ml plgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 1 ug/ml of IgA was preincubated with 10 ul of periplasmic extract containing Nanobody of the different clones and a control with only IgA (high control). The IgA was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, IgA binding was revealed using a rabbit- anti-IgA secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control).

In order to determine IgA competition efficiency of IgA competitive Nanobodies clones of the binding assay were tested in an ELISA competition assay setup. In short, 5 ug/ml plgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 1 ug/ml of IgA was preincubated with a dilution series of purified Nanobody and a control with only IgA (high control). The IgA was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, IgA binding was revealed using a rabbit-anti-IgA secondary antibody (Serotec cat# AHP525H), which was after a wash step detected with a HRP-conjugated donkey-anti- rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control) and two Nanobodies that can bind to pϊgR but do not compete for IgA binding. The results confirm that clones 1D2, 1D7, 1E7, 4Bl 1 and 4D9 have an antagonistic effect on IgA binding to plgR. 1D2 and 4B11 are inferior in this to the other clones.

In order to determine binding specificity to plgR in cells Nanobody 4Bl 1 was tested in an immunofluorescence setup (adapted from Klapisz 2002). In short, MDCK cells overexpressing human plgR were grown on glass cover slips and free binding sites were blocked using precooled 4% Marvel in PBS at 4C. Next, 3uM of purified Nanobody in 50 ul 2% Marvel PBS was allowed to bind to the cells at 4C. After incubation and a wash step, cells were fixed using 4% paraformaldehyde. Nanobody binding was revealed using a mouse- anti-myc secondary antibody, which was after a wash step detected with a Cy2-conjugated donkey-anti-mouse antibody. For reference nuclei were stained using DAPI. Fluorescence signal was detected under an epi fluorescence microscope (Lei ca) attached to a cooled CCD camera (Micromax, Princeton Instruments). The pictures were taken using Metamorph and the final figures were obtained using the NIH Image and Adobe Photoshop programs. Pictures show that Nanobody 4Bl 1, 1D2 and 1E7 can bind to human plgR in a cellular environment.

Example 2,8: Nanobodies are able to bind the fapIgR in its native form

In order to check whether Nanobodies are able to bind the hpIgR in its native form, an immunoprecipitation experiment was performed. Nanobodies, containing a His-tag, were allowed to bind to hpIgR in cell lysates and fished out with talon beads. The hpIgR was detected on blot with a-hSC and DAG-PO. As a control the VHHs bound to the beads were detected with a-myc and DAM-PO. The result of this immunoprecipitation experiment clearly shows that the VHHs: 1D2, 4B11, 4B7 and 1D7 are able to bind to hpIgR in cell lysates. The receptor could be detected in the four lanes containing Iy sates with the hpIgR binding VHHs. Empty talon beads and nanobodies directed against the EGF receptor were not able to detect the receptor and the receptor was also not detected in lysates of untransfected MDCK cells. The lysate control shows that the nanobodies are able to enrich for this receptor out of cell lysate. The binding of the Nanobodies to the talon beads was checked and this shows that indeed all lanes contained beads with bound VHHs, except for the empty beads and the VHHs were also not present in the cell lysate.

Example 23: t raiascytosis c^paeity of the Nanobodies Nanobody-ϊodination with lodogen method

Glass tubes were coated with lodogen by dissolving 10 mg lodogen in 10 ml chloroform in the fume hood and letting 100 μl of the solution evaporate in a clean tube with a gentle stream OfN₂. The tube was twisted while applying a constant air flow on the tube.

Next nanobodies were iodinated by washing the Iodogen-coated glass tube twice with 5 ml buffer I (0.5 M K-phosphate buffer. pH 7.5). The labeling reaction was started by pipetting in the glass tube, 5 μl Na¹²⁵I (0.5 mCi) and 50 ug of nanobody in a total volume of 300 ul buffer I. (50 ug samples of purified nanobodies 1-7 (resp.) 1D2, 1D7, 1E7, 4B7, 4B11, 49F5 (anti- FcgRl) and 500 ug IgA.)

This mixture was left to react for 10 minutes, gently mixed a few times and the reaction slopped by pipetting the mixture to a clean eppendorf tube and adding 200 μl buffer I to the mixture.

Labeled nanobody and free label were separated on a AGl column. 500 ul fraction were collected in buffer II (0.25 M K-phosphate buffer, pH 7.5, 1% BSA).

Subsequently, the specific activity of^I25I-nanobody was determined by TCA precipitation of 5 ul of each fraction. AU samples showed less then 5% of free label. Binding assay

MDCK cells expressing plgR or wildtype MDCK cells were grown in a 12 wells plate. Cells were blocked by addition of Marvel up to a final concentration of 2% Marvel for 1 hour. 3.2 nM of labeled nanobody was allowed to bind to MDCK cells bearing plgR or wild type (-) cells. Binding occurred for 1 hour and cells were washed and Iysed before counting. Negative control is 49F5 anti-FcgRI nanobody. The results show that the labeled nanobodies were able to specifically bind to plgR on cells. Labelled IgA was not able to bind (Figure 8). Competition assay

MDCK cells expressing plgR were grown in a 12 wells plate. Cells were blocked by addition of Marvel up to a final concentration of 2% marvel for 1 hour. 3.2 nM of labeled nanobody was allowed to bind to MDCK cells bearing plgR in the absence or presence of 1 uM of unlabeled nanobody. Binding occurred for 1 hour and cells were washed and lysed before counting. Negative control is 49F5 anti-FcgRI nanobody. The results show that the labeled nanobodies were able to specifically bind to plgR on cells, because binding can be competed off with "cold" nanobody.

Transcytosis assays

In short, transwell system with collagen-coated filter (Corning) were seeded with 100000 cells per well and cells were allowed to polarize for four days. A day before the experiment the medium was changed Into CO2 independent medium. Wells containing confluent polarized MDCK cells expressing plgR were blocked by addition of Marvel to a final concentration of 1 % of Marvel. Labelled nanobodies were added to the medium in the basolateral or apical chamber. Samples were taken from the other chamber after set time points and radioactivity was measured in a gamma counter.

In the first experiment 48 or 4.8 nM of nanobody 1D2 and 4B7 and the negative control nanobody was allowed to transcytose over MDCK wildtype and MDCK plgR from basolateral to apical for 4.5 hours. The results are shown in Figure 10. This shows that 1D2 and 4B7 transcytose over MDCK cells using plgR from basolateral to apical and that 4.8 nM nanobody is preferred for this assay, as shown by the low background.

Subsequently four nanobodies (1D2, 1E7, 4B7 and 4BI 1) were allowed to transcytose over MDCK wildtype and MDCK plgR from basolateral to apical for 23 hours (Figure 1 1). Again all four nanobodies show significant transcytosis over MDCK plgR (left-hand side of panel) compared to transcytosis over MDCK WT (right-hand side of panel). The somewhat higher signal in the MDCK WT with 4B 11 was due to some contamination that happened when talcing the 5 hour sample. In a further experminet (Figure 29), all plgR VHHs were almost not translocated over MDCK wildtype cells similarly to a negative control VHH. The negative control VHH showed similar translocation over MDCK cells overexpressing plgR. In contrast, the plgR nanobodies were significantly translocated over MDCK cells expressing pϊgR. Calculating from the specific activity of the labeled nanobodies approximately 10-30 ng of nanobody is transcytosed per 24 hours. This indicates that the nanobodies undergo plgR mediated transcytosis from basolateral to apical.

To check the competition potential of IgA the same experiment was conducted with IgA present in the basolateral compartment. A 250x molar excess of IgA was added to the basolateral compartment just prior to application of the labeled nanobodies. The results show that IgA is able to compete for binding to the receptor which leads to lower amounts of nanobody being transcytosed from basolateral to apical (Figure 12).

To study the transcytosis from apical to basolateral the four nanobodies (1D2, 1E7, 4B7 and 4Bl 1) (4.8 nM) were applied apically and samples were taken basolaterally. To avoid re- transcytosis from basolateral back to apical an excess of unlabelled nanobody to the basolateral compartment (marked with +). The results show that three of the four nanobodies show significant transcytosis compared to the negative control nanobody (Figure 13). In a further experiment (as above in this paragraph), figure 30 shows that at least two times as much VHH was transported as compared to VHH 49F6. Calculating from the specific activity of the labeled nanobodies approximately 0.5 ng of nanobody is transcytosed per 5 hours. This implicates that VHH are able to be translocated from apica] to basolateral using plgR.

ELISA

In order to check cross-reactivity with mouse plgR an ELISA was performed. In short, 2 ug/ml mouse or human plgR (R&D) was coated o/n in PBS. 1 uM and 10 nM nanobody was allowed to bind. Bound nanobody was detected with 9E10 mouse-anti-myc (Figure 14).

Materials and assays:

Generation of phages:

PCR fragments of the different nanobodies (i.e. 1D2, 4D9, 4Bl 1, 4B7, 4G6, 1F7, 2D2, 2F3, 2B4) were generated with primers M13rev and GENE3 primer (LGTC, The Netherlands), from the masterplate. PCR fragments were reamplified with primers M13rev and MPE25 (LGTC. The Netherlands) and subsequently cut with Sfil and BstEII. These fragments were ligated into ρax51 for periplasmic expression of nanobody and in pax50 for phage production (both vectors are described in WO2008074868). The identity of the clones was checked via per and sequencing and glycerol stocks were made of the clones in pax50 and pax51 by addition of 20% glycerol (final concentration).

From the glycerol stock overnight cultures were made in 5 ml LB 2%glu/amp. The next day these cultures were diluted 1:100 in 50 ml 2TY 2% glu/amp and grown for three hours untill OD 0.6-0.9. Subsequently, to 5 ml of culture 5 ul of helperphage VCSM13 (10 12 pfu) was added and left at 37C for 30 min without shaking. After infection, cells were spun down and redissolved in 2TY/amp/kaπ medium and phages were allowed to be produced overnight.

Subsequently cells were spun down 15 min. @ 4600 rpm. and 40 ml of supernatant was added to 10 ml ice cold 20% PEG6000/2.5M NaCl₅ incubate 30-60 min. on ice. Spin precipitated phages 15 min. @ 4600 rpm, discard as much supernatant as possible (e.g.: keep tubes upside down a while). Resuspend pellet into ImI PBS. Spin 5 min., max. speed. Add the supernatant to 250ul 20% PEG6000/2 5M NaQ, incubate 10 min. on ice. Spin 5 min., max. speed, remove supernatant. Spin 5 min. max. speed, remove supernatant and resuspend pellet into ImI PBS. Add 500 ul glycerol and phages were stored at -80C.

a-SC is goat anti-secretory component antibody recognizing plgR (prod nr. S1640_; sigma)

anti-ml3-hrp is from Amersham.

ELISA (Binding of phages to hpIgR)

Wells were coated with the ectodomain of hplgR (R&D systems). Binding was detected with anti-ml3 conjugated to HRP. As a negative control binding of an aspecific phage and of anti- mi 3-HRP without phages was tested. Coating was checked with a-SC. Yellow bars represent a dilution of 1 μl phage containing solution in I ml. Green bars represent a dilution of 10 nl in 1 ml. Transcytosis assay

First we wanted to show that the phages were able to transcytose across the cells in the most common direction, from basolateral to apical,

Lucifer Yellow was added to the basolateral chamber one hour before the experiment as a control for monolayer integrity and a-specific transport. The concentration of Lucifer yellow (LY) in the apical chamber was determined by measuring fluorescence. The apical LY samples showed no leakage or a-specific transport. After the experiment the monolayer was fixed and stained with dapi. Filters were examined to check the integrity of the monolayers and they appeared to be intact. Phages were added to the basolateral chamber of the Transwell-system and allowed to transcytose for 5 hours. At multiple time points samples were taken from the apical chamber and the total amount of transcytosed phages was determined.

Monoclonal phages 1D2 and 4B7 were able to transcytose across the monolayer of MDCK cells bearing the hpIgR, whereas they could not cross the MDCK cells without hpIgR. Also an irrelevant phage expressing GST-binding V_RHS did not transcytose across transfected or untransfecled cells.

The transcytosis assay is performed with fully polarized MDCK cells, seeded on 1 cm², 0.4 μm collagen-coated PTFE filters (Costar). The filters were washed with DMEM-buffered medium without phenolred (Invitrogen). 2G0μ! medium was added to the apical chamber, 300 μl medium with 2% marvel to the basolateral chamber. Lucifer yellow (25μg/ml) was added to the basolateral chamber to control for paracellular leakage. After 60 minutes an apical sample was taken to determine the concentration of Lucifer yellow (LY). Phages were pre- incubated for 30 minutes with 2% marvel in DMEM w/o phenol red. Subsequently, 10 μl phages of 1 x 10⁶ TU/ml was added to basolateral chamber or 10 μl phages of 1 x 10⁸ TU/ml was added to the apical chamber. The filters were incubated for 5 hours or overnight. Samples were taken from the apical chamber every hour for 5 hours and after 23 hours. The number of transcytosed phages on either side was determined as E. coli TgI transforming units (TUs), which are the phages that are able to infect an E. coli cell and thereby providing this E. coli with resistance to ampiciline. Each colony that grows on a LB ampiciline plate represents one transcytosed phage that was able to infect an E. coli cell. LY concentrations were quantified by measuring fluorescence between 510-520 nm (excitated at 450 nm). After the experiment the cells on the filters were fixed and stained with dapi, embedded in 4μl mowiol+PPD and allowed to dry for 2 hours. Monolayers on the embedded filters were checked for integrity with fluorescent microscopy. When Luficer Yellow and or the dapi staining showed any abnormalities transcytosis results were not included in this report.

Results: Elisa: Figure 31

Transcytosis of cargo from basolateral to apical: VHH (^nanobodies) transcytosing from basolateral to apical could be used as a carrier to deliver therapeutics to the mucosal tissues. To determine the ability to bring cargo from the blood to the mucosal tissues VHH were tested on their ability to carry phages from the basolateral to the apical surface of MDCK cells using pϊgR. Therefore, 10⁶TU of lD2-phage or 4B7-phage were used in the transwell system in a transcytosis assay. Apical samples were taken after each hour up to five hours and allowed to infect bacteria, where after colony forming units (CFU) were determined. Phages coupled to 1D2 and phages coupled to 4B7 were both translocated to the apical side of MDCK cells overexpressing hpϊgR (see figure 32). These phages were not able to cross MDCK cells without overexpression of plgR. Phages coupled to VHH-anti-GST were not able to cross both polarized epithelial cells. This indicates that VHH binding to hpIgR are able to translocate cargo from the basolateral side of polarized epithelial cells using hpIgR.

Example 3: Nanobodjgg_and Naraobødy constructs against mouse EpoR, GHR and moiase Leptiα Receptor

General The erythropoietin receptor (EpoR) and the growth hormone receptor (GHR) belong to the cytokine receptor type I superfamiϊy for which signaling is known to be triggered by Hgand- induced receptor homodimerization and mediated by cytoplasmic protein tyrosine kinases of the Jak family (Watowich SS., Activation of erythropoietin signaling by receptor dimerization. The International Journal of Biochemistry & Cell biology (1999). 31: 1075- 1088; Frank SJ. Receptor dimerization in GH and Erythropoietin action- It takes two to tango, but how? Endocrinology (2002). 143: 2-10; Brooks AJ₅ Wooh JW, Tunny KA and Waters MJ. Growth hormone receptor; mechanism of action. The International Journal of Biochemistry & Cell biology (2007). Doi: 10.1016/1 Bioceϊ, 2007.07.008).

In the case of the EpoR. upon binding of erythropoietin (Epo), receptor dimerization and activation of the signal transduction pathway lead to erythroid cell survival, proliferation and differentiation. The GHR dimerization and signaling induced by the growth hormone is the key regulator of postnatal growth and has important actions on metabolism, reproductive, gastrointestinal cardiovascular, hepto-biliary and renal systems (Brooks et al₅ 2007, supra).

Because of the existence of many clinical situations where the circulating red blood cell levels are reduced provoking anemia, some efforts have been made to develop stable and potent erythropoietin mimetic peptides (EMPs) that activate the receptor by dimerization and thus mimic Epo action. Some bivalent monoclonal antibodies have been described as EpoR agonist since they are capable of forming receptor dimers and stimulate cell proliferation in EpoR-expressing cells, while monovalent Fab fragments fail. For the related GH receptor, a variety of agonist monoclonal antibodies have been also reported.

Structure of EpoR and GHR and interaction with natural ligands

The EpoR and GHR, as other members of the cytokine receptor type I superfamily, are cell surface proteins composed of an NH₂-termina! ligand binding domain, a COOH-terminal cytoplasmic region and a single membrane-spanning domain. Conserved features of the extracellular domain include two pairs of cysteine residues and a -WSXWS'^* motif with characteristic spacing. While receptor dimerization is a common activation mechanism for this family of cytokine receptors there seem to be small differences between the protein folding pathways and/or three-dimensional structures of individual receptors which dictate their potential to be covalently dimerized by disulfide bridges. The classical model for activation of GHR is described as the formation of a ligand-receptor complex made up of one GH molecule and two GHR (GH: 2GHR). One of the monomer receptors binds with a strong affinity to site 1 of the GH followed by the weaker site 2 binding to the second receptor (Watowich, 1999, supra; Brooks et al, 2007, supra). Other studies revealed that the receptor can be found as a dimer on the surface of the cell in the absence of GH leading to a paradigm shift whereby most evidences support a model of GH binding to a constitutively homodimerised GHR which causes the recognition of the intracellular domains resulting in the activation of the signal transduction (Brooks et al., 2007, supra; Waters MJ, Hoang HN, Pelekanos RA and Brown RJ. New insights into growth hormone action. Journal of Molecular Endocrinology (2006). 36: 1-7). This current model of signaling also applies to the closely related EpoR (Watowich, 1999, supra). Early studies of the EpoR/Epo complex suggested a 1:1 stoichiometry, although later studies demonstrated a 2:1 stoichiometry showing two binding sites of Epo for the extracellular domain of the receptor. Interaction of the first site is of high affinity (dissociation constant 1 nM) while the second binding interaction is much weaker (1 uM). There are also some reports that evidence for preformed dimers of EpoR before ligand activation. In that case the binding of Epo changes the orientation of the two receptor subunits, transmitting a conformational change through the transmembrane domains leading to activation of JAK2 kinase and induction of proliferation and survival signals.

Disease relation Reduction of red blood cell levels by a failure in the Epo synthesis provoking anemia is associated to many pathological conditions including chronic renal failure, malignancy or the effects of chemotherapy used to treat cancer. HIV and rheumatoid arthritis (Watowich, 1999, supra), So Epo is used normally therapeutically administered either by intravenous or subcutaneous injection. However the fact that Epo is large glycoproteins has a negative impact on the cost of the manufacture and on the mode of delivery of this therapeutic agent. Therefore the development of new molecules that can mimic the Epo trough interaction with EpoR is clearly a need.

GH has been of significant scientific interest for decades because of its capacity to dramatically change physiological growth parameters. GH has been used for the treatment of adults with GH deficiency and conditions such as Turner's syndrome, Prader-Willie syndrome, intrauterine growth restriction and chronic renal failure. Mutations in the GHR have been described as the cause of the Laron Syndrome that is characterised by severe postnatal growth retardation. Example 3.1: Identification of potential agonist nanobodies binding EpoR

Members:

Families of binders: members: -I 261-B5, 261 -G9, 261-H3, 261-F3, 261-D11, 261-E10 -II 261-H10 -III 261-A8 -IV 261-F8 -V 260-F6 -VI 260-D5 -VII 261-C6 -VIII 260-E8

-IX 260-E5

All families except family V were shown Io inhibit mouse Epo binding to mouse EpoR-Fc.

Sequence alignments of mouse EpoR-Fc binding nanobodies (Figure 15).

Example 3.2: Animal Im munizations

Two llamas (215 and 216) were immunized, according to standard protocols, with 6 boosts of a cocktail 152 containing the recombinant mouse EpoR/Fc Chimera (R&D Systems Cat No 1390-ER, Lot GWROl 0707A). This recombinant protein was obtained from a DNA sequence encoding the signal peptide from human CD33 (Met 1-Ala 16) joined with the extracellular domain of mouse erythropoietin receptor (Ala 17-Pro 249) and fused to the Fc region of human IgGl (Pro 100-Lys 330) via a peptide linker. Blood were collected from these animals 4 and 8 days after boost 6.

Example 3.3: LibrarLgonstruction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA were extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

ExgMBl^A4i_SeIegtions of phage displaying mEpoR binding Nanobodies Phage libraries 215 and 216 were used for selections on recombinant mouse EpoR/Fc Chimera (R&D Systems Cat No 1390-ER). rm EpoR/Fc was coated independently at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control)on Maxisorp 96 well niicrotiter plates (Nunc). To minimize the number of phage binding to the Fc-portion of EpoR/Fc the phage were pre- incubated with 100 ug/ml human IgG (SIGMA, Cat No 14506, Lot 047K7635) and 100 ug/ml of recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06). Following incubation with the phage libraries and extensive washing, bound phage were totally eluted with trypsin. Individual colonies of E. coli TGl infected with the obtained eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (perϊplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 3.5: Screening for mEpoR binding Nanobodies

In order to determine binding specificity to mEpoR, the clones were tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to EpoR/Fc Chimera (R&D Systems Cat No 1390-ER) were tested. Shortly, 0.2 ug/ml receptor were immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS were allowed to bind to the immobilized antigen. After incubation and various wash steps, phage binding were revealed using a HRP- conjugated monoclonal-anti-Ml 3 antibody (Gentaur Cat# 27942101). Binding specificity was determined based on OD values compared to controls having received no phage and to controls where in a similar ELISA binding assay the same monoclonal phage were tested for binding to 0.2 ug/ml of immbolized human IgG and Tie2/Fc. Figure 16 shows a selection of clones binding to mEpoR-Fc.

bind mEpoR

It has been reported that activation of the EpoR requires dimerization of the receptor. In order to determine whether the mEpoR binding monovalent nanobodies could also bind the receptor in a bivalent format to get EpoR dimerization, pseudobivaJent nanobodies were generated by incubating purified mEpoR nanobodies with an anti-myc antibody (Roche, Cat no 1667203). Nanobody binding ELISAs to mEpoR were performed using pseudobivalent and monovalent nanobodies as follows:

1 ug/ml receptor was immobilized on Maxisorp ELlSA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. In parallel, 10 and 100 iiM of the Nanobodies were incubated with 1 ug/ml of anti myc antibody in 100 ul 2% Marvel/PBS for one hour. These pseudobivalent nanbodies were then incubated 1 hour with the coated mEpoR and after some washings, bound nanobodies were revealed using a anti mouse-AP (SIGMA, A2429) . In a separated plate monovalent nanobodies ( 10 and 100 nM in 2%

Marvel/PBS) were added to immobilized EpoR for an hour. Then 1 ug/ml of anti-myc was added to the plate wells for another hour and after some washing bound monovalent nanobodies was revealed using a anti mouse- AP (SIGMA, A2429).

Binding specificity was determined based on OD values compared to controls having received no nanobody or an irrelevant one.

Figure 17 shows results of this assay using a selection of clones binding to mEpoR.

All these nanobodies seem to be good candidates for bivalent nanobody constructs to get EpoR dimerization and therefore activation. Nevertheless, it is to notice that among these mEpoR nanobodies 260-F6 seems to be low affinity one since binding was only shown at 100 nM and in the bivalent format. The same binding improvement effect was observed for 261- C6 and 260-E5 for both concentrations, 100 and 10 nM. The rest of the nanobodies seem to bind with the same affinity in the mono and bivalent format. Exainglje 3Jι__.Sereemngjor Nanobodies competing Epo-EpoR interaction. Clones tested positive in the EpoR binding assay and belonging to different families were screened for their ability to block rnEpo binding to mEpoR-Fc. For this, Nanobody- containing periplasmic extracts (P.E.) were used in an ELISA-based Iigand competition setup. In short, 0.75 ug/rnl mouse erythropoietin (mEpo) (R&D Systems Cat No 959-ME/CF Lot No EUP0207051) was coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml mEpoR-Fc was incubated with 10 ul of periplasmic extract containing nanobody of the different clones in 100 ul 2% Marvel/PBS₌ After 1 hour, the receptor-Nanobody pre-mixes were incubated 1 hour with the coated Iigand, Bound mEopR-Fc was detected using HRP~conjugated goat anti-human IgG (Jackson Immunore search, Cat # 109-035-098). Blocking activity was determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P. E., had been added.

Figure 18 shows results of this blocking assay using a selection of clones binding to mEpoR.

Example 3.8: Determining Epo-EpoR interaction blocking effieieney by titration of purified Nanobody

In order to determine the receptor blocking efficiency of clones tested positive for Epo competition, a dilution series of purified Nanobodies were tested in the ELISA-based Iigand competition setup.

In short, 0.75 ug/ml mouse erythropoietin (mEpo) (R&D Systems Cat No 959-ME/CF Lot No EUP0207051) was coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml mEpoR-Fc was incubated with a dilution series of purified Nanobodies in 2% Marvel/PBS (100 ul). After 1 hour, the receptor- Nanobody pre-mixes were incubated for another hour with the coated Iigand. Bound mEopR- Fc was detected using HRP-conjugated goat anti-human IgG (Jackson Immunoresearch, Cat # 109-035-098). Blocking activity was determined as loss of OD signal, as compared to wells where no nanobody or irrelevant nanobody was added. Figure 19 shows the results of this assay.

From these results it seems that all these mEpoR nanobodies , except 260-F6, are blocking the interaction with mEpo showing low nM IC50 values. Example 3.9: Construction of bivalent mEpoR nanobodies

The strategy of the generation of bi- or multivalent Nanobodies has previously been successfully applied to target other proteins and may result in a significant increase in avidity (J. MoI. Biol (2004) 341, 161-169).

Bivalent mEpoR nanobodies were constructed using a PCR-based method where two copies of the same nanobody were connected by a 20 Gly~Ser linker (four repetitive GGGGS).

Example 3.10: Titration of binding of monovalent and bivalent nanobodies to mEpoR

In order to determine whether the bivalent mEpoR nanobodies were binding to mEpoR and to compare this binding with the correspondent monovalent nanobody, an ELISA was performed using the following protein and protocol:

Recombinat mouse EpoR/Fc chimera obtained from a DNA sequence encoding the signal peptide from human CD33 and joined with the extracellular domain of mouse erythropoietin receptor (Ala25-Pro249; Accession # P14753). This was fused to the Fc region of human IgGl via a peptide linker and expressed in a mouse myeloma cell line, NSO (R&D System Cat No l390-ER)

Briefly, 1 ug/ml of receptor was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Monovalent and bivalent purified nanobodies (0-10 nM in 2% Marvel/PBS) were added to immobilized EpoR for an hour. Then 1 ug/ml of anti-myc was added to the washed plate wells for another hour and after some washing bound monovalent nanobodies was revealed using an anti mouse- AP (SIGMA, A2429).

Binding specificity was determined based on OD values compared to controls having received no nanobody or an irrelevant one.

Figure 33 shows results of this assay using a selection of clones binding to mEpoR. From these results it is possible to estimate roughly the ΪC50 binding values of the bivalents nanobodies. In general all the monovalent Nbs (except 260-F6) show IC50 values between 1.25 and 2.5 nM. Bivalents Nbs 261-F3, 261-H3, 261-H10 and 261-F8 show IC50 values around 300-600 pM. In the case of bivalents Nbs 261-A8 and 260-F6 this value increases up to around 1 nM. When compared to the monovalents, the bivalent nanobodies seem to show an improvement in affinity of 2-5 folds and even higher in the case of 260-F6. For that particular nanohody, the monovalent format might have a very low affinity for EpoR since no binding was detected using 100 nM of the nanobody. Binding of 260-F6 was only shown by phage ELISA, where phage surface "multidisplay" of the nanobody contributes to increase the avidity for the coated receptor.

Example 3.11: Testing Screened non-competing Nanobodies (non-neutralizing) for

EpoR agonism using MAPPIT (J. Tavernier et al., MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells, Clin. Exp. Allergy 2002; 32: 1397-1404). Reference is made to the assay described in J, Tavernier et al, supra. In short, a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric "bait" construct, e.g. a construct wherein the extracellular domain of the leptin receptor (LR) is replaced by the murine (see vecior pCEL If in Tavernier's publication above) or human (see vector pSELl same publication) ligand binding extracellular Epo-R gene; b) infection of cells stably expressing the "bait" with above identified bivalent Nanobody constructs; and c) stimulation and selection using puromycin with results in surviving clones that express the agonistically acting bivalent Nanobody construct (confirmation that bivalent Nanobody construct is indeed acting agonistically).

Example 3.12: Cell assay to demonstrate agonist or antagonist activity of mEpoR

Nanobidies using HCD57 cells.

The mouse HCD57 erythro-leukemia cell line proliferates when Epo is added to the cells.

Upon redrawal of Epo cells undergo extensive apoptosis. Epo-induced cell proliferation can be measured 3H-TdR incorporation or by cell viability MTT staining (MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is cleaved by an enzyme in the respiration chain in the mitochondria if the cell is viable, generating MTT formazan a dark blue, highly visible product. Wash the microcarriers with PBS. Add a solution of MTT (5 mg/ml in PBS, 1 part MTT solution to 10 parts microcarriers). Incubate for 45 minutes at 37 °C. Observe the cells with a light or phase-contrast microscope. Apoptosis of HCD57 cells can be detected by using flow cytometry analysis of propidium iodide-stained cells (Jacobs- Helber, S. M., Ryan. J. J.. and Sawyer, S. T. (2000) Blood 96, 933-940) or DNA fragmentation analysis. Epo receptor agonist Nanobodies when added to the Epo starved HCD57 cells are expected to reduce the number of apoptotic cells and to increase proliferation. Epo receptor antagonist Nanobodies when added to Epo stimulated HCD57 cells are expected to inhibit TdR incorporation, hence proliferation and to increase the number of apoptotic cells.

Exampie 3.13: Identification of agoaist nanobodies binding GHR

Members:

Families of binders: members

=1 268-H5 -II 268-B7 -III 268-B12 -IV 267-B3 -VI 267-HlO -VII 268-F12 -VTTT 268-H7

Sequence alignments are shown in Figure 20.

Example 3.14: Animal Immunizations Two llamas (215 and 216) were immunized, according to standard protocols, with 6 boosts of a cocktail 152 containing the recombinant mouse GHRTFc Chimera (R&D Systems Cat No 1360-GR, Lot No GHQ030707A). This recombinant protein was obtained from a DNA sequence encoding the extracellular domain of mouse growth hormone receptor (Thr 25-Asp 273), fused to the signal peptide of human CD33 (Met 1- Ala 16) at the amino-termus and to the Fc region of human IgGl (Pro 10G-Lys 330) at the C-terminus. Blood was collected from these animals 4 and 8 days after boost 6.

Example 3.15: Library construction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoϊl-Hypaque according to the manufacturer's instructions. Next, total RNA were extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 3.16: Selections of phage displaying mGHR binding Namobodies Phage libraries 215 and 216 were used for selections on recombinant mouse GHR/Fc Chimera (R&D Systems Cat No 1360-GR). rm GHR/Fc was coated independently at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control)on Maxisorp 96 well microtiter plates (Nunc). To minimize the number of phage binding to the Fc-portion of EpoR/Fc the phage were pre-incubated with 100 ug/ml human IgG (SIGMA₅ Cat No 14506, Lot 047K7635) and 100 ug/ml of recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-Tl, Lot No BKC06). Following incubation with the phage libraries and extensive washing, bound phages were totally eluted with trypsin. The eluted phage were amplified and applied in a second round of selection on 0.5 ug/mL 0.05 ug/ml, 0.005 ug/ml and 0 ug/mi (control) immobilized GHR/Fc. Individual colonies of E. coli TGl infected with the obtained eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein). Optionally, the Phage libraries may be pre-incubated with jejunal or gastric fluid prior Io in order to select for protease-resistant Nanobodies. Based on preliminary reports in one arm a GI fluid concentration is chosen that results in. a decrease in antigen binding capacity in phage ELlSA to 10% of an untreated control, In another arm. the Phage libraries are selected for EpoR binding in the presence of jejunal or gastric fluid (again pre-incubated and not pre-incubated).

Example 3.17: Screening for mGHR binding Nanobodies

In order to determine binding specificity to mGHR, the clones were tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to GHR/Fc Chimera (R&D Systems Cat No 1360-GR) were tested. Shortly, 0.2 ug/ml receptor were immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS were allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding were revealed using a HRP- conjugated monoclonal-anti-M13 antibody (Gentaur Cat# 27942101). Binding specificity was determined based on OD values compared to controls having received no phage and to controls where in a similar ELISA binding assay the same monoclonal phage were tested for binding to 0.2 ug/ml of immbolized human IgG and Tie2/Fc. Figure 21 shows a selection of clones binding to mGHR-Fc.

Example 3.18: Screening for Nanobodies competing GH-GHR interaetioa or not competing GH-GHR

Clones tested positive in the GHR binding assay (including clones selected for protease resistancy) are screened for their ability to block GH binding to GHR/Fc. For this, positive binding GHR phages are used in an ELISA-based ligand competition setup. 10 ul of supernatant from the monoclonal phage inductions of the different positives clones is mixed with increasing amounts of GH and added to 96 well Maxisorp microliter plates (Nunc) coated with GHR. After incubation and washing steps, phage binding is revealed using a HRP-conjugated monoclonal-anti-MIS antibody (Gentaur Cat# 27942101). Binding specificity is determined based on OD values compared to controls having received no GH and/or no phage. The same kind of competition assay could be performed using Nanobody- containing periplasmic extracts (P. E.) instead, of phage and detecting with a mouse anti-myc antibody and a anti mouse-HRP antibody.

Clones tested positive in the GHR binding assay (including clones selected for protease resistancy) are screened for their ability not to block GH binding to GHR/Fc. For this, positive binding GHR phage are used in an ELlSA-based ligand competition setup. 10 ul of supernatant from the monoclonal phage inductions of the different positives clones is mixed with increasing amounts of GH and added to 96 well M axisorp microtiter plates (Nunc) coated with GHR. After incubation, eluted phage containing non-neutralizing Nanobodies are further analyzed e.g. in BioCore experiments and verified whether indeed they are non- neutralizing Nanobodies. In fact, these non-neutralizing Nanobodies are used preferably for the construction of agonistic construct comprising e.g. 2 GHR non-neutralizing Nanobodies. Thus, e.g. various constructs are generated comprising 2 GHR non-neutralizing Nanobodies that are identified above, e.g. linked by a 9 GIy linker (see e.g. WO 2007/104529).

Example 3.19: Testing Screened ison-competing Nanobodies (non-neutralizing) for identifying GHR agonism using MAPPIT (J. Tavernier et a!.. MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells, Clin. Exp. Allergy 2002; 32: 1397-1404).

Reference is made to the assay described in J. Tavernier et al, supra. In short, a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric "bait" construct, e.g. a construct wherein the extracellular domain of the leptin receptor (LR) is replaced by the murine (see vector pCEL If in Tavernier' s publication above) or human (see vector pSELl same publication) ligand binding extracellular Epo-R gene; b) infection of cells stably expressing the "bait" with above identified bivalent Nanobody constructs; and c) stimulation and selection using puromycin with results in surviving clones that express the agonistically acting bivalent Nanobody construct (confirmation that bivalent Nanobody construct is indeed acting agonistically). Example 3.20: In vivo model to test systemic delivery In Vivo Model for Epo-R agonist read out (Spiekermann et al, 2002, supra). Female BALB/c mice 4-6 wk of age and control C57BL/6 mice from e.g. The Jackson Laboratory are maintained under pathogen-free conditions. Mice are anaesthetized with e.g. ϊsoflurane by inhalation and the different Compounds of the invention (e.g. as disclosed above, e.g. construct comprising 2 anti-mouse non-neutralizing Epo-R Nanobodies (e.g. with 9 GIy linker), optionally comprising a pH independent or pH dependent anti-mouse FcRn or plgR Nanobody (i.e. binding at gut pH, about pH 6, but released at blood pH7 or more) are injected intraperitoneally, gauged into small intestine, fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart), or administered orally by a enterically coated capsule for mouse consumption, e.g. capsule from example 32. Mice are killed by CO₂ inhalation 8 h or 4 d later and whole blood is obtained by cardiac puncture.

Flow Cytometric Analysis. Whole blood samples from above are added to e.g. ReticOne Reagent according to the manufacturer's instructions. Flow cytometry is performed with e.g. a Coulter Epics XL machine. Acquisition parameters are calibrated each time by e.g. Retic-Cal Biological Calibration and Retic-C Cell Control 40,000 total events in the red blood ceil gate are acquired and analyzed with ReticOne automated software for percentage of reticulocytes (all materials e.g. from Beckman Coulter). Increase in number of reticulocytes in blood is indicative of functional delivery of Epo-R agonists into body, i.e. systemic delivery.

Note: For Epo-R dimerization, the agonistically acting Nanobody construct may have a Koff equal or lower than 1 nM since interaction of the first site of Epo for EpoR is of high affinity (dissociation constant 1 nM) while the second binding interaction is much weaker (1 uM). To be determined e.g. in BioCore experiments. In vivo model for GHR agonist read out -In vivo assay to test GHR agonists (Wang et al., 1996 Molecular and Cellular Endocrinology, Volume 116, Issue 2, 5 February 1996, Pages 223-226). -Studies on promotion of animal growth using GH deficient hypophysectomized rats.

Similarly as above, mice are anaesthetized with e.g. Isoflurane by inhalation and the different Compounds of the invention (e.g. as disclosed above, e.g. construct comprising 2 anti-mouse non-neutralizing GHR Nanobodies (e.g. with 9 GIy linker), optionally comprising a pH independent or pH dependent anti-mouse FcRn or plgR Nanobody (i.e. binding at gut pH, about pH 6, but released at blood pH7 or more) are a) injected intraperitoneally, b) gauged into small intestine, c) fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart), or d) administered orally by e.g. a enterically coated capsule acceptable for mouse consumption, e.g. capsule from example 32. Mice growth is monitored.

Increase in growth is indicative of a systemically delivered GHR agonist.

Note: For GHR, the agonistically acting Nanobody construct against GHR from above may have a Koff equal or lower than 0.3 nM since interaction of GH to GHR-dimer was reported to be in the range of 0.3 nM (Cunningham et al, 1989, Science 244:1081-1085.). To be determined e.g. in BioCore experiments.

Example 3,21: Oral/tntragastrie administration of Nanobodies directed against the moasejeptin receptor.

Leptin plays a crucial role in the regulation of body weight. The Nanobody 4.10-Albl (SEQ TD NO: 1 13) is a leptin receptor antagonist. Daily treatment during 7 days of mice with this Nanobody results in increased body weight and increased serum levels of insulin and leptin (see Figures 34, 35, 36) 7 daily injections of 4.10~Albl, followed by ConA administration on day 7 (200 ug/mouse) causes increased serum concentrations of ALT/AST at 24 h post injection, demonstrating that 4.10-Albl aggravates ConA induced hepatitis (Figure 37). Nine to 10 week old C57B16 mice could be orally or intragastrically administered with Nanobodies (400-1000 ug/day during 7 days; volume 100-500 ul). Possible degradation of Nanobodies in the gastrointestinal tract by proteases at low pH (stomach) could be prevented temporarily by the use of soybean trypsin inhibitor; an antacid, for example sodium bicarbonate (Hifumi et al., 2008); or Bictra (sodium citrate) (Warny et al., 1999). An acid antisecretory therapy with the proton pump inhibitors like omeprazole could also prevent degradation (Lyerly et al., 1991; Kelly et al., 1997; Warny et al., 1999). Finally enteric encapsulation of Nanobodies could also be used to protect the Nanobodies against degradation. Such capsules for example coated with two layers of cellulose acetate phytate can remain intact for at least 2 h at pH = 1 to 2 but release their contents within 30 min at pH - 6 (Kelly et al., 1997). Also the use of a feeding formula could enhance the protection against degradation in the stomach and intestine (Lyeriy et al, 1991, Zeitlin et al., 1999). To study the effect of the Nanobody treatment, the body weight of the mice could be determined daily. In addition blood could be collected and the serum concentrations of insulin and leptin could be determined. An increase in body weight and insulin and leptin serum concentrations or increased AST/ ALT levels after CONA injection would demonstrate that the orally/intragastrically administered leptin receptor Nanobody has become available systemically.

Orai/tistragastrie administration of Nanobodies directed against the mouse Epo reeeptor.

A similar in vivo setup to demonstrate oral to systemic delivery could be performed with Nanobodies that agonize (or antagonize) the mouse Epo receptor. Here it is expected that an oral to systemic availability of an agonistic Epo receptor Nanobody would increase the reticulocyte fraction in blood. The oral to systemic circulation of an antagonist Epo receptor Nanobody is expected to reduce the reticulocyte fraction. To determine the reticulocyte fraction, whole blood can be obtained by cardiac puncture and whole blood samples can be added to ReticOne Reagent according to the manufacturer's instructions. Flow cytometry can be performed with a Coulter Epics XL machine (Spiekemiarm et aL, J. Exp. Med. Volume 196, Number 3, August 5, 2002 303-310)

Example 4: Identification glconditional serum albnim.ro specific nanobodies Example 4.1: ImmumizatioB

After approval of the Ethical Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), 2 llamas (117, 118) were alternately immunized with 6 intramuscular injections at weekly interval with human serum albumin and a mixture of mouse serum albumin, cynomolgus serum albumin and baboon serum albumin, according to standard protocols.

Example 4«2:_Library eomstraetioB

When an appropriate immune response was induced in llama, four days after the last antigen injection, a 150 ml blood sample is collected and peripheral blood lymphocytes (PBLs) were purified by a density gradient centrifugation on Ficoll-Paque™ according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage is prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored at 4⁰C for further use.

Example 43LS^ecJjn£χep_ertg|res for binding to serum albumin.

In a first selection, human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-welI plates (Nunc₅ Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates were blocked with 4% Marvel in PBS for 2h at RT. After 3 washes with PBST, phages were added in 4% Marvel/PBS and incubated for Ih at RT. Following extensive washing, bound phage was eluted with 0.1 M tiiethanolamine (TEA) and neutralized with IM Tris-HCl pH 7.5.

Example 4.4; Selecting repertoires for conditional binding to serum albumin.

To enrich for conditional binders, said binders with a pH sensitive interaction, phage libraries were incubated with antigen at physiological pH and eluted at acidic pH as follows.

In a first selection, human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates are blocked with 4% Marvel in PBS pH 7.3 for 2h at RT. After 5 washes with PBS/0.05% Tween20 (PBST) pH 7.3, phages were added in 2% Marvel/PBS pH 7.3 and incubated for 2h at RT. Unbound phages were removed by 10 washes with PBST pH7.3, followed by 2 washes with PBS pH5.8. Bound phage was eluted with PBS ρH5.8 for 30 min at RT and neutralized with IM Tris-HCl pH 7.5.

In a second selection, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvell/CPA buffer (10 mM sodium citrate + 10 mM sodium phosphate + 10 mM sodium acetate + 115 mM NaCl) adjusted to pH 7.3. Unbound phages were removed by 10 washes with CPA/0.05%Tween20 (CPAT) pH7.3, followed by 2 washes with CPAT pH5.8. Bound phage was eluted with CPA pH5.8 for 30 min at RT and neutralized with IM Tris- HCl pH 7. In a third selection strategy, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvel 1/CP A pH5.8. Unbound phages are removed by 10 washes with CPAT pH5.8, followed by 2 washes with CPA pH 7,3. Bound phage was eluted with Img/ml trypsin/CPA pH 7.3 for 30 min at RT.

In a fourth selection strategy, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvell/PBS pH5.8. Unbound phages are removed by 10 washes with PBST pH5.8, followed by 2 washes with PBS pH 7.3. Bound phage was eluted with img/ml trypsin/CPA pH 7.3 for 30 min at RT.

In all selections, enrichment was observed. The output from each selection was re-cloned as a pool into the expression vector pAXSl. Colonies were picked and grown in 96 deep-well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ~ 80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant).

Example 4.5: Library evaluation by ELISA.

Periplasmic extracts of individual Nanobodies were screened for albumin specificity by ELISA on solid phase coated human serum albumin. Detection of Nanobody fragments bound to immobilized human serum albumin was carried out using a biotinylated mouse anti- Ms antibody (Serotec MCAl 396B) detected with Streptavidin-HRP (DakoCytomation #P0397). The signal was developed by adding TMB substrate solution (Pierce 34021) and detected at a wavelength of 450 nm. A high hit rate of positive clones can already be obtained after panning round 1.

Example 4.6: Selection for conditional or pH-sensitive binding of Nanobodies to albumin by ELISA.

To enrich for conditional binders, said binders with a pH sensitive interaction, phage libraries may be incubated with antigen at physiological pH and eluted at acidic pH as follows.

In a first selection strategy, human serum albumin (Sigma A-8763) is coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates are blocked with 4% Marvel in PBS pH 7.3 for 2h at RT. After 5 washes with PBS/0.05% Tween20 (PBST) pH 7.3, phages were added in 2% Marvel/PBS pH 7.3 and incubated for 2h at RT. Unbound phages were removed by 10 washes with PBST pH7.3, followed by 2 washes with PBS pH5.8. Bound phage was eluted with PBS pH5.8 for 30 min at RT and neutralized with 1 M Tris-HCl pH 7.5.

In a second selection strategy, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvell/CPA buffer (10 mM sodium citrate + 10 niM sodium phosphate + 10 niM sodium acetate + 1 15 mM NaC!) adjusted to pH 7.3. Unbound phages were removed by 10 washes with CPA/0.05%Tween20 (CPAT) pH7.3, followed by 2 washes with CPAT pH5,8. Bound phage was eluted with CPA pH5,8 for 30 min at RT and neutralized with IM Tris-HCl pH 7.

In a third selection strategy, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvell/CPA pH5.8. Unbound phages are removed by 10 washes with CPAT pH5.8, followed by 2 washes with CPA pH 7.3. Bound phage is eluted with lmg/ml trypsin/CPA pH 7.3 for 30 min at RT.

In a fourth selection strategy, phage libraries were incubated for 2h at RT with human serum albumin in 2% Marvell/PBS pH5.8. Unbound phages are removed by 10 washes with PBST pH5.8, followed by 2 washes with PBS pH 7.3. Bound phage was eluted with lmg/ml trypsin/CPA pH 7.3 for 30 min at RT.

In all selections, enrichment is observed. The output from each selection was re-cloned as a pool e.g. into the expression vector pAX51. Colonies are picked and grown in 96 deep-well plates (1ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ~ 80 μl) are prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). Example 4.7: Screening of Nanobody repertoire for the occurrence of a pH-sensitive interaction via surface plasmon resonance (BIAcore).

Human serum albumin was immobilized on a CM5 sensor chip surface via amine coupling using NHS/EDC for activation, and ethanolamine for deactivation (Biacore amine coupling kit).

Approximately IOOORU of human serum albumin was immobilized. Experiments were performed at 25 °C . The buffers used for the pH dependent binding of Nanobodieε to albumin (Biacore) are as follows: 10mM Sodium citrate (Na₃C₆H₅O₇) + 1OmM Sodium phosphate (Na₂HPO₄) + 1OmM Sodium Acetate (CH₃COONa) + 115mM NaCL This mixture is brought to pH7, pH6 and pH5 by adding HCl or NaOH (dependent on the pH of the mixture measured).

Periplasmic extracts were diluted in running buffers of pH7, pH6 and pH5. The samples were injected for lmin at a flow rate of 45ul/min over the activated and reference surfaces. Those surfaces were regenerated with a 3s pulse of glycine-HCl pHl.5 + 0.1% P20. Evaluation was done using Biacore TlOO evaluation software.

The off rate of different Nanobodies at pH7 and pH5 is documented in Table E-I . The majority of the Nanobodies (4A2, 4A6, 4B5, 4B6, 4B8, 4C3, 4C4, 4C5, 4C8, 4C9, 4D3, 4D4, 4D 7 ad 4D10 have a faster off rate at pH 5 compared with pH 7 (2-6 fold difference in off rate). The Nanobody 4A9 has a slower off-rate at pH 5 compared to pH 7 (0.54 fold difference in off rate). For other Nanobodies including 4C12, 4Bl, 4B10, IL6R202, Alb-8, and 4D5, binding to antigen does not change at different pH.

Direct screening of nanobody repertoires for conditional binding to antigen can thus be used.

Table B-7: Off rate (determined by Biacore) of different Nanobodies at pH7 and pH5 is documented (see also WO2008043822 for sequence information and methods of making below sequences and below Table B-8)

Example 4.8: Screening for conditional binding of Nanobodies by ELISA To screen Nanobodies for their conditional binding to albumin, a binding ELISA can also be performed with two representative conditions, pH 5.8 and pH7.3 and the relative binding strength determined. Maxisorb micro titer plates (Nunc, Article No. 430341) were coated overnight at 4⁰C with 100 μl of a 1 μg/ml solution human serum albumin in bicarbonate buffer (50 mM, pH 9.6). After coating, the plates were washed three times with PBS containing 0.05% Tween20 (PBST) and blocked for 2 hours at room temperature (RT) with PBS containing 2% Marvel (PBSM). After the blocking step, the coated plates were washed 2 times with PBST pH 5.8, and a ten-fold dilution aliquot of each periplasmic sample in PBSM pH5.8 (lOOμl) is transferred to the coated plates and allowed to bind for 1 hour at RT. After sample incubation, the plates were washed five times with PBST and incubated for 1 hour at RT with 100 μl of a 1 :1000 dilution of mouse anti~myc antibody in 2% PBSM. After 1 hour at RT, the plates were washed five times with PBST and incubated with 100 μl of a 1 : 1000 dilution of a goat anti-mouse antibody conjugated with horseradish peroxidase. After 1 hour, plates were washed five times with PBST and incubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20 minutes, the reaction was stopped with 100 μl H₂SO₄. The absorbance of each well was measured at 450 ran.

92 periplasmic extracts for each of the conditional selection strategies described herein, are analyzed in this ELISA. Table B-6 depicts the result for Nanobodies that conditionally bind to human serum albumin at neutral pH, i.e. pH 7.4, but not to acidic, i.e. pH 5.8. Table B-7 depicts the results for Nanobodies that conditionally bind to human serum albumin at acidic pH, i.e. pH 5.8, but not to neutral pH, i.e. pH 7.4.

Upon 1 round of selection on human serum albumin, followed by total elution, Nanobodies are identified that either conditionally bind to albumin at acidic pH (n= 16) or at neutral pH (n-19). Driving the selection conditions towards conditional binding, results in a higher ratio of conditionally binding nanobodies (n=23).

Example 5; Analysis of effect of conditional binding on pharmacokinetic behaviour of Example 5.1: Construction of bi- or mtiltispecific nanobody format Bispecific nanobodies are e.g. generated by construction of a C-terminal pH dependent FcRn binding Nanobody, a 9 amino acid Gly/Ser linker (e.g. GGGGSGGGS) and an N-terminal anti-target Nanobody, e.g. an N-terminal polypeptide with 2 Nanobodies against EPO-R functioning as agonist on human and/or murine EPO-R or an N-terminal polypeptide with 2 Nanobodies against EPO-R functioning as agonist on human or murine GHR. These constructs may be expressed in E.coli as c-myc, Hϊsό-tagged proteins and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

Example 5.2: Retention of conditional binding upon foriMttm^Bto_.jiijiti§iedfie format

The conditional pH- binding properties of the anti-FcRn or plgR Nanobody or dAbs within the multispecific nanobody formats (e.g. are evaluated via surface plasmon resonance (BIAcore), e.g. a conditional binder as disclosed in this application is linked to one or more nanobody or dAbs binding to one or more protein target(s), e.g. is linked to 2 Nanobodies directed against Epo-R or GHR. Cross-reactivity to cynomolgus serum albumin is also assessed. Human and cynomolgus FcRn or plgR are immobilized on a CMS sensor chip surface via amine coupling using NHS/EDC for activation and ethanolamine for deactivation (Biacore amine coupling kit).

Experiments are performed at 25⁰C. The buffers used for the pH dependent binding of

Nanobodies to FcRn or plgR (Biacore) are as follows: 1OmM Sodium citrate (Na^CβΗ.sOη) + 1 OmM Sodium phosphate (Na₂HPO₄) + 1OmM Sodium Acetate (CH₃COONa) + 115mM NaCl. This mixture is brought to pH7, pHό and pH5 by adding HCl or NaOH (dependent on the pH of the mixture measured).

Purified Polypeptides are diluted in running buffers of pH7, pH6 and pH5. The samples are injected for lmin at a flow rate of 45ul/min over the activated and reference surfaces. Those surfaces are regenerated with a 3s pulse of glycine-HCl pH1.5 + 0.1% P20. Evaluation is done using Biacore TlOO evaluation software.

Example 5.3: Pharmacokinetic profile of mailtisperific Bamobody formats in eynorooigns monkey delivered by Lv. injection A pharmacokinetic study is conducted in cynomolgus monkeys. A Compound of the invention (e.g. Epo-R or GHR agonistic bivalent polypeptide with FcRn or plgR pH dependent binding block, i.e. 2 Epo-R or 2 GHR binding blocks linked via a 9 amino acid Gly/Ser linker to each other and a conditional FcRn or plgR binding block, again linked e.g. via a 9 amino acid Gly/Ser linker) is administered intravenously by bolus injection (1.0 ml/kg, approximately 30 sec) in the vena cephalica of the left or right arm to obtain a dose of 2.0 mg/kg. The Nanobody concentration in the plasma samples is determined via ELISA.

The concentration in the plasma samples is determined as follows: Maxisorb micro titer plates (Nunc, Article No. 430341) are coated overnight at 4⁰C with 100 μl of a 5 μg/ml solution of the Compound of the invention in bicarbonate buffer (50 mM, pH 9.6). After coating, the plates are washed three times with PBS containing 0.1% Tween20 and blocked for 2 hours at room temperature (RT) with PBS containing 1% casein (250 μl/well). Plasma samples and serial dilutions of polypeptide- standards (spiked in 100% pooled blank cynomolgus plasma) are diluted in PBS in a separate non-coated plate (Nunc, Article No. 249944) to obtain the desired concentration/dilution in a final sample matrix consisting of 10% pooled cynomolgus plasma in PBS. All pre-dilutions are incubated for 30 minutes at RT in the non-coated plate. After the blocking step, the coated plates are washed three times (PBS containing 0.1% Tween20), and an aliquot of each sample dilution (lOOμl) is transferred to the coated plates and allowed to bind for 1 hour at RT. After sample incubation, the plates are washed three times (PBS containing 0.1% Tween20) and incubated for 1 hour at RT with 100 μl of a 100 ng/ml solution of sIL6R in PBS (Peprotech, Article No. 20006R). After 1 hour at RT, the plates are washed three times (PBS containing 0.1% Tween20) and incubated with 100 μl of a 250 ng/ml solution of a biotinylated polyclonal anti-Compound of the invention in PBS containing 1% casein (R&D systems, Article No. BAF227). After incubation for 30 minutes (RT), plates are washed three times (PBS containing 0.1% Tween20) and incubated for 30 minutes (RT) with 100 μl of a 1/5000 dilution (in PBS containing 1% casein) of streptavidine conjugated with horseradish peroxidase (DaktoCytomation, Article No. P0397). After 30 minutes, plates are washed three times (PBS containing 0.1% Tween20) and incubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20 minutes, the reaction is stopped with 100 μl HCl (IN). The absorbance of each well is measured at 450 run (Tecan Sunrise spectrophotometer), and corrected for absorbance at 620 ran. This assay measures free Compound of the invention as well as Compound of the invention bound Polypeptide of the Invention. Concentration in each plasma sample is determined based on a sigmoid standard curve with variable slope of the respective Polypeptide of the Invention,

Each individual plasma sample is analyzed in two independent assays and an average plasma concentration is calculated for pharmacokinetic data analysis.

All parameters are calculated with two-compartmental modeling, with elimination from the central compartment.

Example 6: Preparation of various pharmaceutical orally deliverable compositions a) Capsules comprising the Compounds of the invention - preferably enteric coated capsules For the purposes of illustrating this invention in e.g. a monkey or mouse, the enteric coating material is selected from HPMC-AS (pH 5.5), CAT (pH 5.5) and Eudragit L (pH 5.5), most preferably Eudragit L (pH 5.5),

For use in the human, the enteric coating material preferably may be one which will provide for release of polypeptide at about pH 6.0-6.5 such as, for example, CAP and HPMC-AS.

The enterocoating is carried out by methods known per se in the art, e.g., according to Remington Pharmaceutical Sciences, p. 1614-1615 (1975, 15th Ed., Mack Pub. Co.) and Theory and Practice of Industrial Pharmacy, Lackman, Liberman & Caning, p. 116-117, 371- 374 (1976, 2nd Ed.). The enteric micro-encapsulation process is also known (Theory and Practice of Industrial Pharmacy ibid, pp. 420-438). See also Remington Pharmaceutical Sciences, p. 1637 (1985, 17th Ed., Mack Pub. Co.). Typically, the amount of enteric coating material used preferably is in the range about 10-20 mg per 500 cm.sup.2 of surface area of capsule or tablet, especially of capsule as produced in the actual examples described herein below. The amount of enteric coating material broadly is in the range of about 1-5000 mg/capsule, more preferably about 10-1000 mg/capsule, most preferably about 50-100 mg/capsule.

A solution comprising the Compounds of the invention (e.g. the herein described examples, e.g. agonistic GHR or EpoR polypeptides (i.e. bispecific construct comprising 2 Nanobodies against GHR or EpoR including construct additionally comprising a FcRn or plgR binding Nanobodies (preferably pH dependent binding, e.g. binding at pH 6 or less but not or to a much lower extend at pH 7 and more)) is filled up into enteric coated capsules and used within a short time, e.g. within a week or day for the in vivo experiment as e.g. presented in the below examples,

A liquid formulation will generally consist of a solution or suspension containing the biologically active polypeptide, e.g. the Compound of the invention and optionally protease inhibitor(s) filled into a pharmaceutically acceptable capsule for example, a standard or conventional hard gelatin capsule and the filled capsule will be coated, e.g. as described above. The solution or suspension which is filled into such capsule will generally consist of the biologically active Compound of the invention and protease inhibitor(s) dissolved or suspended in any pharmaceutically acceptable liquid carrier such as, for example, a sterile aqueous carrier or water-miscible solvents such as, for example, ethanol, glycerin, propylene glycol and sorbitol, or mixtures of any of the foregoing.

Preferred aspects:

I) A pharmaceutical composition for oral or nasal administration comprising a therapeutically effective amount of a compound comprising one or more single variable domain(s) directed against FcRn, plgR or Vit B 12 receptor and optionally a pharmaceutically acceptable enteric coating, preferably FcRn. more preferably directed against FcRn binding site without inlerefering IgG and/or serum albumin binding to FcRn.

2) The composition according to aspect I₃ wherein said compound comprises or essentially consists of a single Nanobody, domain antibody, single domain antibody or "dAb", preferably a Nanobody.

3) The composition according to aspect 1, wherein said compound comprises or essentially consists of at least two Nanobodies, domain antibodies, single domain antibodies or

"dAbs", preferably a Nanobody.

4) The composition according to aspect 3, wherein said compound comprises or essentially consists of at least one Nanobody, domain antibody, single domain antibody or "dAb" against one epitope, antigen, target, protein or polypeptide and at least one other

Nanobody, domain antibody, single domain antibody or "dAb" directed against another epitope, antigen, target, protein or polypeptide.

5) The composition according to aspect 1, wherein said compound comprises one or more Nanobodies, domain antibodies, single domain antibodies or "dAbs" linked to one or more amino acid, sequence that provides an increased half-life following delivery to the subject.

6) The composition according to aspect 5, wherein said one or more amino acid sequence is a Nanobody, a domain antibody, a single domain antibody or a "dAb", preferably a

Nanobody. 7) The composition according to aspect 6, wherein said one or more amino acid sequence is directed against a serum protein.

8) The composition according to aspect L wherein said polypeptide comprises one or more Nanobodies, domain antibodies, single domain antibodies or "dAbs" linked to one or more amino acid sequences that allow the resulting polypeptide to cross the epithelial membrane of the gut.

9) The composition according to aspect 8, wherein said one or more amino acid sequences is a Nanobody, a domain antibody, a single domain antibody or a "dAb" , preferably a

Nanobody.

10) The composition according to aspect 1, wherein said compound comprises a therapeutic polypeptide or agent linked to a Nanobody, a domain antibody, a single domain antibody or a "dAb" directed against an epithelial trans-membrane protein on the intestinal membrane.

11) The composition according to any of aspects 1 to 10, wherein said Nanobody, domain antibody, single domain antibody or "dAb" is derived from a V_H or V_HH-

12) The composition according to aspect 11, wherein said Nanobody, domain antibody, single domain antibody or "dAb" is a humanized V_HH or a camelized V_H.

13) The composition according to any of aspect 1 to 12, which, upon oral or nasal administration to a subject, induces a systemic therapeutic or biological response in said subject,

14) The composition according to any of aspects 1 to 13, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches the bloodstream with a Cmax of at least 1 ng polypeptide or protein per ml of blood following oral administration of a dose of 5 mg/kg body weight of said polypeptide. 15) The composition according to any of aspects 1 to 14, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches a Cmax in blood of at least 1% of the Cmax that is reached following parenteral administration of the same amount of polypepti de .

16) The composition according to any of aspects 1 to 15, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches the bloodstream with a Tmax of less than 120 minutes following oral administration of said composition to said subject.

17) The composition according to any of aspects 1 to 16, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches the bloodstream with a Cmax of at least 1 ng polypeptide per ml of blood within less than 120 minutes following oral administration of a dose of 5 rag/kg body weight of said polypeptide to said subject.

18) The composition according to any of aspects 1 to 17, wherein, upon oral or nasal administration of said composition to a subject, the AUC for the compound in blood is at least 500 ng/ml/minute polypeptide following oral administration of a dose of 5 mg/kg body weight of said polypeptide to said subject.

19) The composition of any of aspects 1 to 1 8, wherein, upon oral or nasal administration of said composition to a subject, the compound has an absolute and/or relative bioavailability in blood of at least 1%.

20) The composition according to any of aspect 1 to 12, which, upon oral or nasal administration to a subject, induces a therapeutic or biological response in said subject.

21) The composition according to aspect 20, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches a Cmax of at least 1% of the Cmax that is reached following parenteral administration of the same amount of compound. 22) The composition according to any of aspects 20 or 21, wherein, upon oral or nasal administration of said composition to a subject, the compound reaches a Tmax of less than 120 minutes following oral administration of said composition to said subject.

23) The composition of any of aspects 20 to 22, wherein, upon oral or nasal administration of said composition to a subject, the compound has an absolute and/or relative bioavailability of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably 10% in the blood compared to the directly in the blood, injected said compound.

24) The composition of any of aspects 1 to 23, wherein the enteric coating is an anionic polymer and dissolve at ranges from pH 5.5 to pH 7.

25) The composition of any of aspects 1 to 24, wherein the enteric coating is an anionic polymer comprising methacrylic acid and methacrylates and dissolve at ranges from pH 5.5 to pH 7.

26) The composition of any of aspects 1 to 25, additionally comprising a permeability enhancer such as e.g. acyleamitme or N~(5-chlorosalicyloyl)-8-aminocaprylic acid.

27) The composition of any of aspects 1 to 26, additionally comprising protease inhibitor such as e.g. organic acids.

28) The composition of any of aspects 1 to 27, wherein the compound comprises at least one Nanobody.

29) The composition of any of aspects 1 to 28, wherein the compound comprises at least one Llama-derived Nanobody.

30) The composition of any of aspects I to 29, wherein the compound comprises at least one Llama-derived Nanobody which is humanized.

31) The composition of any of aspects 1 to 30, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of FcRn preferably FcRn₅ more preferably directed against extracellular part of a FcRn binding site without interefering IgG and/or serum albumin binding to FcRn.

32) The composition of any of aspects 1 to 29, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR.

33) The composition of any of aspects 1 to 29, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of Vit B 12 receptor.

34) The composition of aspect 31 , wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of FcRn, more preferably directed against extracellular part of a FcRn binding site without interefering IgG and/or serum albumin binding to FcRn, preferably with a Kd of 100 nM, 1OnM, 1 nM, 100 pM or 10 pM, more preferably a Kd of 10 nM or 1 nM, e.g. a Kd of 1O nM.

35) The composition of aspect 32, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR, preferably with a Kd of 100 nM, 1OnM, 1 nM, 100 pM or 10 pM, more preferably a Kd of 10 nM or 1 nM, e.g. a Kd of 10 nM.

36) The composition of aspect 33, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of Vit B 12 receptor, preferably with a Kd of 100 nM, 1OnM, 1 nM, 100 pM or 10 pM, more preferably a Kd of 10 nM or 1 nM, e.g. a Kd of 10 nM.

37) The composition of any of aspect 31 , wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of FcRn and wherein said Nanobody directed against the extracellular part of FcRn is cross-blocked by any FcRn Nanobody of SEQ ID NOs: 1 to 7, more preferably is cross-blocked by any FcRn Nanobody of SEQ ID NOs: 1 to 4.

38) The composition of aspect 31, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of FcRn and wherein said Nanobody directed against the extracellular part of FcRn. more preferably directed against extracellular part of a FcRn binding site without interefering IgG and/or serum albumin binding to FcRn, cross-blocks FcRn Nanobody of SEQ ID NOs: 1 to 13, more preferably SEQ ID NOs: 1 to 7, more preferably SEQ ID NOs: l to 4.

39) The composition of aspect 31, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of FcRn and wherein said Nanobody directed against the extracellular part of FcRn, more preferably directed against extracellular part of a FcRn binding site without interefering IgG and/or serum albumin binding to FcRn, has 70%, 75%, 80%, 85%, 90% sequence identity (measured e.g. by blasting 2 sequences with blastp and scoring matrix BLOSUM62 (Henikoff & Henikoff, 1992)) to FcRn Nanobody of SEQ ID NOs: 1 to 13, more preferably SEQ ID NOs: 1 to 1, even more preferably SEQ ID NOs: 1 to 4.

40) The composition of aspect 32, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR and wherein said Nanobody directed against the extracellular part of plgR is cross-blocked by plgR Nanobody of SEQ ID NOs: 14 to 34.

41) The composition of aspect 30, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR and wherein said Nanobody directed against the extracellular part of plgR cross-blocks plgR Nanobody of SEQ ID NOs: 14 to 34.

42) The composition of aspect 30, wherein the compound comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR and wherein said Nanobody directed against the extracellular part of plgR has 70%, 75%, 80%, 85%, 90% sequence identity (measured e.g. with blast 2 sequences with blastp and scoring matrix BLOSUM62 (Henikoff & Henikoff, 1992)) to plgR Nanobody of SEQ ID NOs: 14 to 34.

43) The composition of any of aspects 31 to 42, wherein the binding of the Nanobody directed against the extracellular part of plgR, FcRn or Vit B12 receptor is pH dependent.

44) The composition of aspect 43, wherein the Nanobody directed against the extracellular part of plgR, FcRn or Vit B12 receptor binds at pH6 or lower, e.g. pH5, to its receptor and does significantly less (e.g. 2, 3, 4, 5, or 10 times or not at all) bind to its receptor at pH7 and higher.

45) The composition of any of aspects 31 to 44, wherein the compound has agonistic properties to the target molecule.

46) The composition of any of aspects 31 to 44, wherein the compound has antagonistic properties to the target molecule.

47) The composition of aspect 45 wherein the compounde is an agonist or antagonist to Epo- R₅ preferably human or mouse Epo-R; or the composition of aspect 45 wherein the compound is an agonist or antagonist to GHR, preferably human or mouse GHR: or composition of aspect 45 wherein the compound is an agonist or antagonist to Leptin Receptor, preferably human or mouse Leptin Receptor.

48) The composition of aspect 45 wherein the compound is an agonist or antagonist to Epo-R, preferably human or mouse Epo-R.

49) The composition of any of aspects 1 to 48 wherein the compound is proteolytically stabilized, e.g. is selected for proteolytic stability.

50) The composition of aspect 49 wherein the proteolytically stabilized properties are resulting from a compound consisting essentially of proteolytically stabilized (e.g. screened for proteolytically stabilized properties) Nanobodies that are e.g. linked with Gly/Ser linkers.

5I) A method for delivering a compound comprising or essentially consisting of at least one Nanobody. a domain antibody, a single domain antibody or "dAb" to the bloodstream of a subject, said method comprising the step of orally or nasally administering a composition according to any of aspects 1 to 50 to said subject.

52) A method for delivering a compound comprising or essentially consisting of a Nanobody, a domain antibody, a single domain antibody or "dAb" to the gut of a subject, said method comprising the step of orally administering a composition according to any of aspects 1 to 50 to said subject.

53) A method for the preparation of a composition according to any of aspects 1 to 50.

54) A method for the prevention and/or treatment of a subject in need of compound comprising or essentially consisting of at least one Nanobody, domain antibody, single domain antibody or "dAb", said method comprising the step of orally or nasally administering to said subject a compound, Nanobody, a domain antibody, a single domain antibody or a "dAb" as described above and/or a composition comprising the same.

55) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a Nanobody, a domain antibody, a single domain antibody or a "dAb" to a subject suffering from said disease or disorder, said method comprising the step of orally or nasally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" as described above, and/or of a composition comprising the same.

56) A method for immunotherapy comprising oral administering to a subject suffering from or at risk of a diseases and/or disorders that can be cured or alleviated by immunotherapy with a Nanobody, a domain antibody, a single domain antibody or a '"dAb", a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" as described above and/or of a composition comprising the same.

57) Use of a Nanobody, a domain antibody, a single domain antibody or a "dAb^*' for the preparation of a composition/compound as described herein for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally or nasally administering to a subject a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein.

58) The composition according to any of aspects 1 to 50, for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Nanobody, a domain antibody, a single domain antibody or a "dAb".

59) A method for the prevention and/or treatment of a subject in need of a compound, a Nanobody, a domain antibody, a single domain antibody or a^udAb" that is directed against a target in the kidney or bladder, said method comprising orally or nasally administering, to said subject a therapeutically effective amount of said, compound , Nanobody, domain antibody, single domain antibody or '"dAb" as described herein and/or of a composition comprising the same.

60) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in the kidney or the bladder, said method comprising orally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb'^" as described herein and/or of a composition comprising the same.

6I) A method for the prevention and/or treatment of a disease or disorder of the kidney or bladder, said method comprising orally administering to said subject a therapeutically effective amount of a compound, a Nanobody, a domain antibody, a single domain antibody or a '"dAb" that is directed against a target in the kidney or the bladder and/or of a composition comprising the same.

62) Use of a compound, aNanobody, a domain antibody, a single domain antibody or a "dAb" directed against a target in the kidney or the bladder for the preparation of a medicament as described herein for the prevention and/or treatment of at least one a disease or disorder of the kidney or bladder.

63) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against a target in the kidney or the bladder for the prevention and/or treatment of a disease or disorder of the kidney or bladder.

64) A method for the prevention and/or treatment of a subject in need of a compound, Nanobody, a domain antibody, a single domain antibody or a '"dAb^"' that is directed against a target in the lung, said method comprising orally or nasally administering, to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or '"dAb" as described herein, and/or of a composition comprising the same.

65) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in the lung, said method comprising orally or nasally administering to said subject a therapeutically effective amount of said compound.

Nanobody, domain antibody, single domain antibody or "dAb" as described herein and/or of a composition comprising the same.

66) A method for the prevention and/or treatment of a disease or disorder of the lung, said method comprising orally or nasally administering to said subject a therapeutically effective amount of a compound, aNanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in the lung, said method comprising orally or nasally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" as described herein and/or of a composition comprising the same.

67) Use of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" directed against a target in the lung for the preparation of a medicament said method comprising orally or nasally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" as described herein for the prevention and/or treatment of at least one disease or disorder of the lung.

68) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against a target in the lung for the prevention and/or treatment of at least one disease or disorder of the lung.

69) A method for the prevention and/or treatment of a subject in need of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target on a tumor cell, said method comprising orally or nasally administering, to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" as described herein and/or of a composition comprising the same.

70) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target on a tumor cell, said method comprising orally or nasally administering to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same.

7I) A method for the prevention and/or treatment of a tumor related disease or disorder, said method comprising orally administering to said subject a therapeutically effective amount of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against a target on a tumor and/or of a composition comprising the same.

72) Use of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" described herein directed against a target on a tumor for the prevention and/or treatment of at least one tumor related disease or disorder.

73) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against a target on a tumor for the prevention and/or treatment of at least one tumor related disease or disorder.

74) A method for the prevention and/or treatment of a subject in need of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in gut, said method comprising orally administering, to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same.

75) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, a Nanobody, a domain antibody, a single domain antibody or a

"dAb" as described herein that is directed against a target in the gut, said method comprising orally administering to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same.

76) A method for the prevention and/or treatment of a disease or disorder of the gut (such as intestinally located inflammatory diseases such as IBD or Crohn's disease.

77) A method for the prevention and/or treatment of a subject in need of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein that is directed against TNF, said method comprising orally administering, to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same. 78) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against TNF, said method comprising orally administering to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same.

79) A method for the prevention and/or treatment of a disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease), said method comprising orally administering to said subject a therapeutically effective amount of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein that is directed against TNF and/or of a composition comprising the sam e .

80) Use of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against TNF for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease).

81) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against TNF for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease).

82) A method for the prevention and/or treatment of a subject in need of a compound, a Nanobody, a domain antibody, a single domain antibody or a '"dAb" as described herein and that is directed against vWF, said method comprising orally or nasally administering, to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same. 83) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against vWF, said method comprising orally or nasally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" as described herein and/or of a composition comprising the same.

84) A method for the prevention and/or treatment of a disease or disorder related to platelet- mediated aggregation (such as e.g. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endartereciomy, carotid artery stenosis, critical limb ischaemia, cardioemboiism, peripheral vascular disease, restenosis and myocardial infarction), said method comprising orally or nasally administering to said subject a therapeutically effective amount of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against vWF and/or of a composition comprising the same.

85) Use of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein directed against vWF for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy , carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction).

86) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against vWF for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g. the formation of a non-occhisive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased, arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction).

87) A method for the prevention and/or treatment of a subject in need of a compound, Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering, to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same.

88) A method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering to said subject a therapeutically effective amount of said compound, Nanobody, domain antibody, single domain antibody or "dAb" and/or of a composition comprising the same. 89) A method for the prevention and/or treatment of a disease or disorder associated with IL- 6R, IL-6 and/or with the IL-6/IL-6R complex (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis,

Kaposi's sarcoma, AlDS-related lymphoma, inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE). multiple sclerosis, Castlemaxrs disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus), said method comprising orally or nasaly administering to said subject a therapeutically effective amount of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex and/or of a composition comprising the same.

90) Use of a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein and that is directed against IL-6, ΪL-6R and/or IL-6/IL-6R complex for the prevention and/or treatment of at least one disease or disorder associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex, (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus).

91 ) The composition according to any of aspects 1 to 50, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is directed against IL-6, IL-6R and/or IL-6/ΪL-6R complex for the prevention and/or treatment of at least one disease or disorder associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman^'s disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus).

92) A method for the prevention and/or treatment of an acute disorder or disease, said method comprising orally or nasally administering to said subject a therapeutically effective amount of a compound, a Nanobody,, a domain antibody, a single domain antibody or a "dAb" as described herein and that is capable of alleviating the symptoms of or curing said disorder or disease.

93) A method for selecting compounds, Nanobodies. domain antibodies, single domain antibodies or "dAbs" directed against an epithelial irans-membrane protein, wherein said Nanobodies, domain antibodies, single domain antibodies or "dAbs cross the membrane upon binding to said epithelial trans-membrane protein, said method comprising the step of panning epithelial trans-membrane protein-displaying membranes with a phage library (naϊve or immune) of Nanobodies, domain antibodies, single domain antibodies or "dAbs" and selecting for membrane crossing Nanobodies, domain antibodies, single domain antibodies or "dAbs" by recovering the transported phage from the membrane.

94) Diagnostic method or drug monitoring method comprising the step of orally or nasally administering to a subject a compound, Nanobody, a domain antibody, a single domain antibody or a "dAb" as described herein or a composition comprising the same and detecting said compound, Nanobody, domain antibody, single domain antibody or "dAb".

95) Method according to aspect 94, wherein said compound, Nanobody, domain antibody, single domain antibody or "dAb" is detected in situ. 96) A compound of formula I

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

in which any of FRl to FR4 is an amino acid sequence of framework regions 1 to 4 as e.g. described herein for single variable domains, and in which any of CDRl to CDR3 is an amino acid sequence of the complementarity determining regions 1 to 3 as e.g. described herein for singe variable domains, and in which optionally X and Y refer to a further unit that comprises one or more other groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers; and wherein said compound is directed against a member of the group consisting of pigR, FcRn and Vit B 12 receptor, preferably human pigR, human FcRn. and human Vit B 12 receptor, more preferably human pigR and FcRn, even more preferably human FcRn, even more preferably a binding site to human FcRn not interefering with human serum albumin and/or human IgG, e.g. IgGl .

97) A compound of formula I

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

and in which optionally X and Y refer to a further unit that comprises one or more other or same groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers; and in which any of CDRΪ , CDR2 and CDR3 is selected from the group consisting of a) amino acid sequences with SEQ ID NO: 1 to 34, more preferably SEQ ID NO: 1 to 7, even more preferably SEQ ID NO: 1 to 4, as defined in Table I₅ wherein the framework regions are indicated with XXX and wherein CDRl is represented by the first group of amino acid sequence after the first framework region, CDR2 is represented by the second group of amino acid sequence after the second framework region, and CDR3 is represented by the third group of amino acid sequence after the third framework region; or any of CDRl, CDR2 and CDR3 is selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the CDRs as shown in SEQ ID NO: 1 to 34 and wherein the amino acid sequences of the framework regions (indicated with XXX) are not taken into account for identity calculation purposes; and wherein optionally said compound has a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human pϊgR and FcRn, even more preferably human FcRn of 10^"5 to 10^{" 12} moles/litre or less, and preferably 10^" to 10^-1 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or said compound has a rate of association (k_on-rate) to a member of the group consisting of pϊgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of between 10² M^" V¹ to about 10⁷ M^-1S^-1, preferably between 10^J M^-1S^-1 and 10⁷ M^-1S^-1, more preferably between 10 M^'V¹ and 10⁷ M^-1S^-1, such as between 10³ M^-1S⁴ and 10⁷ M^'V¹; or said compound has a rate of dissociation (k_Off rate) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn between Is^-1 and 10^"6 s^-1, preferably between 10^"2 s^~l and IQ^"6 s^-1, more preferably between 10^" s^"! and 10^" s^" , such as between 10^" s^" and 10^" s^" ; or said compound has an affinity to a member of the group consisting of pϊgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably a binding site to human FcRn not interefering with human serum albumin and/or human IgG, e.g. IgGl, of less than 500 nM, preferably less than 200 nM, more preferably less than IO nM, such as less than 500 pM.

98) A compound according to aspects 96 or 97, wherein said amino acid sequence is at least an immunoglobulin sequence.

99) A compound according to aspects 96 to 98, wherein said amino acid sequence is at least a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi- synthetic immunoglobulin sequence.

100) A compound according to aspects 96 to 99, wherein said amino acid sequence is at least a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

101) A compound according to aspects 96 to 100, wherein said amino acid sequence is at least a Jight chain variable domain sequence (e.g. a V_L-sequence); or a heavy chain variable domain sequence (e.g. a Vn-sequence).

102) A compound according to aspects 96 to 101 , wherein said amino acid sequence is at least a heavy chain variable domain sequence that is derived from a conventional four- chain antibody or that is a heavy chain variable domain sequence that is derived from heavy chain antibody.

103) A compound according to aspects 96 to 102, wherein said amino acid sequence is at least a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody (including but not limited to a V_HH sequence).

104) A compound according to aspects 96 to 103, wherein said amino acid sequence is at least a Nanobody.

105) A compound according to aspects 96 to 104, wherein said amino acid sequence is at least a Nanobody that a) has 70%, more preferably 75%. even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the CDRs of at least one of the amino acid sequences of SEQ ΪD NO: 1 to 34, more preferably SEQ ID NO: 1 to 7, even more preferably SEQ ID NO: 1 to 4; and in which: b) preferably 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 2; and c) optionally said compound has a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn. even more preferably human FcRn, even more preferably a binding site to human FcRn not interfering with human serum albumin and/or human IgG, e.g. IgGl₁ of IG^"5 to 10^'12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably IQ^"8 to 10^-12 moles/litre or less; or said compound has a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR,

FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn. even more preferably human FcRn, even more preferably a binding site to human FcRn not interfering with human serum albumin and/or human IgG, e.g. IgGl , of between 10 NT's^-1 to about 10⁷ M^'V¹, preferably between 10³ M^-1S^-1 and IG⁷ M^"V\ more preferably between 10⁴ IvT¹S^"' and 10⁷ M⁴S^-1, such as between 10⁵ M^-1S^'1 and 10⁷ M^{' 1}S^-1; or said compound has a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably a binding site to human FcRn not interefering with human serum albumin and/or human IgG, e.g. IgGl, between Is^" and 10^" s^" , preferably between 10^"2 s^-1 and 10^"6 s^-1, more preferably between 10° s^-1 and 10^"6 s^-1, such as between 10^"4 s^-1 and 10^" s^-1; or said compound has an affinity a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably a binding site to human FcRn not interefering with human serum albumin and/or human IgG, e.g. IgGL of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

106) A compound according to aspects 96 to 105, wherein any of X and Y or both comprises at least one single variable domain directed against a target molecule such as e.g. human or mouse serum albumin, human or mouse EPO-receptor or a human or mouse growth hormone or a human or mouse leptin receptor, optionally linked by a linker.

107) A compound according to aspects 96 to 106, wherein any of X and Y or both comprises at least one single variable domain directed against a target molecule such as e.g. human or mouse serum albumin, human or mouse EPO-receptor or a human or mouse growth hormone or a human or mouse leptin receptor, optionally linked by a linker, and wherein examples of single variable domains are provided in Table 3, e.g. amino acid sequence with SEQ ID NOs 69 to 11 L and SEQ ID NOs 1 13 to 120, and also amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the amino acid sequences of SEQ ΪD NOs 69 to 111, and SEQ ID NOs 113 to 120. and optionally said compound has a dissociation constant (K_D) to a member of the group consisting of EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R. GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of

10^"D to 10^-12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^" to 10^" moles/litre or less; or a rate of association (k_on-rate) to a member of the group consisting EPO-R₅ GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO- R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of between 10² M^"V to about 10⁷ M^-1S^-1, preferably between 10³ M^'V¹ and IG⁷ M^'V¹, more preferably between 10⁴ M^'V¹ and IQ⁷ 3VfV¹, such as between 10³ M^-1S^-1 and 10⁷ M^{" 1}S^-1 ; or said compounds have a rate of dissociation (k_off rate) to a member of the group consisting of EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R between Is^-1 and 10^"6 s^-1, preferably between 10^~2 s^-1 and I0^"6 s^"!, more preferably between 10^"3 s^-1 and 10^"6 s^-1, such as between 10^"4 s^-1 and 10^"6 s^-1; or said compounds have an affinity a member of the group consisting of EPO-R, GH-R, serum albumin or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R. GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

108) A compound of formula II

X-Z-Y (II) in which Z is an amino acid sequence comprising at least one single variable domain that is directed against a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR. FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl , and human serum albumin binding, and in which optionally any of X and Y is a further unit that comprises one or more other groups, residues, moieties or binding units such as e.g. a nanobody, optionally linked via one or more linkers.

109) Compound according to aspect 108, wherein a) Z is an amino acid sequence comprising at least one single variable domain that is selected from the group consisting of a) amino acid sequences with SEQ ID NO: 35 to 68 and SEQ ID NO: 112 as defined in Table 4; or Z is an amino acid sequence comprising at least one single variable domain that is selected from the group consisting of b) amino acid sequences that have 70%. more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to amino acid sequences with SEQ ID NO: 35 to 68 and SEQ ID NO: 112 as defined in Table 4₅ and wherein optionally b) said compound has a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGL and human serum albumin binding, of 10^"5 to 10^-12 moles/litre or less, and preferably 10^"7 to 10^"i2 moles/litre or less and more preferably 10^"8 to IQ^'12 moles/litre or less; or said compound has a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG. e.g. human IgGl, and human serum albumin binding, of between 10² M^'V¹ to about 10⁷ M⁴S^-1, preferably between 10³ M⁴S^-1 and 10⁷ M^'V

^!, more preferably between 10⁴ IVF¹S^-1 and 10⁷ M^"V^!, such as between 10⁵ M^"V and 10⁷ M^-1S^-1; or said compound has a rate of dissociation (Ic_0Jp rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding, between Is^-1 and 10^"6 s^-1, preferably between 10^" s^-1 and 10^" s^-1, more preferably between 10^"° s^-1 and IG^"6 s^-1, such as between 10^"4 s^"! and 10^"6 s^-1; or said compound has an affinity a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding, of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

110) Compound according to aspects 108 or 109, wherein said amino acid sequence is at least an immunoglobulin sequence.

111) Compound according to aspects 108 to 110, wherein said amino acid sequence is at least a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence,

112) Compound according to aspects 108 to 111, wherein said amino acid sequence is at least a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

113) Compound according to aspects 108 to 112, wherein said amino acid sequence is at least a light chain variable domain sequence (e.g. a V_L-sequence); or a heavy chain variable domain sequence (e.g. a Vi-j-sequence).

114) Compound according to aspects 108 to 113, wherein said amino acid sequence is at least a heavy chain variable domain sequence that is derived from a conventional four- chain antibody or that is a heavy chain variable domain sequence that is derived from heavy chain antibody. 1 15) Compound according to aspects 108 to 114, wherein said amino acid sequence is at least a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody (including but not limited to a V_HH sequence).

1 16) Compound according to aspects 108 to 115, wherein said amino acid sequence is at least a Nanobody.

117) Compound according to aspects 108 to 116, wherein said amino acid sequence is at least a Nanobody that a. has 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the amino acid sequences of SEQ ID NO's: 35 to 68 and SEQ ID NO: 112, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the FR sequences are disregarded; and in which b. preferably 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 2; and c. optionally said compound has a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10° to 10^-12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or said compound has a rate of association (k_on-i^*ate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl , and human serum albumin binding, of between 10² M^~ls^~] to about 10⁷ M^'V¹, preferably between I O³ M¹V¹ and 10⁷ JVT¹S^-1, more preferably between 10⁴ IVfV¹ and 10¹ IVT¹S^-1, such as between 10^J NT's^-1 and 10⁷ M⁴S^-1; or said compound has a rate of dissociation (k_off rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl₅ and human serum albumin binding, between Is^-1 and 10^"6 s^-1, preferably between 10^"2 s^-1 and 10^"6 s^-1, more preferably between ICF s^-1 and iCf⁶ s^-1, such as between 10⁴ s^-1 and 10^"6 s^-1; or said compound has an affinity a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR. FcRn and Vit B 12 receptor, more preferably human plgR and FcRn, even more preferably human

FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGL and human serum albumin binding, of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

118) Compound according to aspects 108 to 117, wherein any of X and Y or both comprises at least one a single variable domain directed against a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker.

119) Compound according to aspects 108 to 118, wherein any of X and Y or both comprises at least one single variable domain directed against a target molecule such as e.g. human or mouse serum albumin, human or mouse EPO-receptor, human or mouse Leptin-receptor, mouse or human IL-6 receptor or a human or mouse growth hormone, optionally linked by a linker, and wherein examples of single variable domains are provided in Table 3, e.g. amino acid sequence with SEQ ID NOs 69 to 1 3 1 and SEQ ID NOs 113 to 120, and also amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the amino acid sequences of SEQ ID NO's: 69 to 1 11 and SEQ ID NOs 113 to 120, and optionally said compound has a dissociation constant

(KD) to a member of the group consisting of EPO-R, GH-R, serum albumin, leptin receptor or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of 10^~5 to 10^-12 moles/litre or less, and preferably 10^"7 to 10^'12 moles/litre or less and more preferably 10^"8 to 10^-12 moles/litre or less; or said compound has a rate of association (k_on-rate) to a member of the group consisting EPO-R, GH-R, serum albumin, leptin receptor or IL-6 receptor, preferably human or mouse EPO-R, GH-R, serum albumin, leptin receptor or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of between IQ² M^-1S^-1 to about 10⁷ M^'V¹, preferably between 10³ M^{' 1}S^'1 and 10⁷ M^-1S^-1, more preferably between IG⁴ rvfV and 10⁷ IvT¹S^*1, such as between 10⁵ M^-1S^-1 and 10⁷ M^" V¹; or said compound has a rate of dissociation (k₀frrate) to a member of the group consisting of EPO-R. GH-R, serum albumin, leptin receptor or

IL»6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R between ls^"! and 1(X⁶ s^"!, preferably between 10^"2 s^"! and IG^"6 s^"!. more preferably between 10^"3 s^'1 and 10^~6 s^-1, such as between 10^"4 s^-1 and IG^"6 s^-1; or said compound has an affinity a member of the group consisting of EPO-R. GH-R, serum albumin, leptin receptor or IL-6 receptor, preferably human or mouse EPO-R. GH-R. serum albumin or IL-6 receptor, more preferably human EPO-R, GH-R, serum albumin or IL-6 receptor, even more preferably mouse or human EPO-R of less than 500 ΏM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

120) A compound of formula 1

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

and in which optionally X and Y refer to a further unit that comprises one or more other or same groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers; and in which any of CDRl, CDR2 and CDR3 is selected from the group consisting of a) amino acid sequences with SEQ ID NO: 1 to 7, even more preferably SEQ ID NO: 1 to 4, as defined in Table 1 , wherein the framework regions are indicated with χ;χχ a^ wherein CDRl is represented by the first group of amino acid sequence after the first framework region, CDR2 is represented by the second group of amino acid sequence after the second framework region, and CDR3 is represented by the third group of amino acid sequence after the third framework region; or any of CDRl, CDR2 and CDR3 Is selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%. even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the CDRs as shown in SEQ ID NO: 1 to 34 and wherein the amino acid sequences of the framework regions (indicated with xxx) are not taken into account for identity calculation purposes; and wherein optionally said compound has a dissociation constant (K_D) to human FCRΓJ of 1(T⁵ to 10^-12 moles/litre or less, and preferably 10^"7 to 10^-12 moles/litre or less and more preferably 10^"s to 10^~12 moles/litre or less; or said compound has a rate of association (k_oπ-rate) to human FcRn of between ] O² M^"V^! to about 10⁷ M^"V^! . preferably between 10³ M^-1 s^-1 and 10⁷ M^-1S^-1 , more preferably between 10⁴ VT¹S^'1 and 10⁷ IVf¹S^-1, such as between 10³ M^-1S^-1 and IG⁷ M^{' 1}S^'1; or said compound has a rate of dissociation (k_Off rate) to human FcRn between Is^-1 and 10^"6 s^-1, preferably between 10^"2 s^-1 and 10^'6 s^-1, more preferably between 10^"3 s^-1 and 10^"6 s^-1, such as between 10^"4 s^-1 and 10^"6 s^"!; or said compound has an affinity human FcRn, of less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM.

121) The compound of aspect 120, wherein the compound shows a significant longer average half life (e.g. more than 1 h, more than 2 h, more than 3h, more than 4 h) than a comparable compound that has a dissociation constant (K_D) to a binding site of human

FcRn that does not interefer with human serum albumin and/or human IgG, i.e. IgGl, binding of 10^" to 10^" moles/litre or more.

122) A compound as herein described, e.g. a compound as described in aspects 96 to 121. for use as a medicament.

123) A pharmaceutical composition for use as a medicament comprising a compound as herein described, e.g. a compound as described in aspects 96 to 121, and optionally pharmaceutically acceptable excipients such as e.g. buffer, stabilizer etc.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described herein. Such equivalents axe intended to be encompassed by the following claims. All references disclosed herein are incorporated by reference in their entirety for the purpose and information indicated in the specification.

Claims

1. A pharmaceutical composition for oral or nasal administration comprising a therapeutically effective amount of a compound comprising one or more single variable domain(s) directed against FcRn, plgR or Vit Bl 2 receptor and optionally a pharmaceutically acceptable enteric coating.

2. A compound of formula I

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

in which FRl to FR4 refer to amino acid sequences of framework regions 1 to 4 as e.g. described herein for single variable domains, respectively, and in which CDRl to CDR3 refer to amino acid sequences of the complementarity determining regions 1 to 3 of single variable domains, and in which optionally X and Y refer to a further unit that comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers; and wherein said compound is directed against a member of the group consisting of plgR, FcRn and Vit B12 receptor.

3. A compound of formula I

X-FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4-Y (I)

in which any of CDRl, CDR2 and CDR3 is selected from the group consisting of a) amino acid sequences with SEQ ID NO: 1 to 34, more preferably SEQ ID NO: 1 to 4, as defined in Table 1 , wherein the framework regions are indicated with XXX and wherein CDRl is represented by the first group of amino acid sequence after the first framework region, CDR2 is represented by the second group of amino acid sequence after the second framework region, and CDR3 is represented by the third group of amino acid sequence after the third framework region; or any of CDRl , CDR2 and CDR3 is selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the CDRs as shown in SEQ ID NO: 1 to 34, more preferably SEQ ID NO: 1 to 4, and wherein the amino acid sequences of the framework regions (indicated with XXX) are not taken into account for identity calculation purposes; and wherein said compound has a dissociation constant (K_D) to a member of the group consisting of human plgR, FcRn and Vit B 12 receptor, of 10^" to 10^" moles/litre or less.

4. A compound of formula II

X-Z-Y (II)

wherein Z is selected from the group consisting of amino acid sequences comprising at least one single variable domain that is directed against a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B 12 receptor- more preferably human plgR and FcRn₅ even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. IgGl , human serum albumin, and in which optionally X and Y refer to a further unit that comprises one or more other groups, residues, moieties or binding units such as e.g. nanobody, optionally linked via one or more linkers.

5. A compound of formula II

X-Z-Y (II)

wherein Z is an amino acid sequence comprising at least one single variable domain that is selected from the group consisting of a) amino acid sequences with SEQ ID NO: 35 to 68; or Z is an amino acid sequence comprising at least one single variable domain that is selected from the group consisting of b) amino acid sequences that have 70%, more preferably 75%, even more preferably 80%, even more preferably 85%. even more preferably 90%, even more preferably 95% identity to amino acid sequences with SEQ ID NO: 35 to 68; and wherein said compound has a dissociation constant (K_D) to a member of the group consisting of human plgR, human FcRn and human Vit B12 receptor of 10^"5 to 10^-12 moles/litre or less,

6. A compound or polypeptide comprising at least a Nanobody wherein said Nanbody a. has 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95% identity to the amino acid sequences of SEQ ID NO's: 35 to 68 and SEQ ID NO: 112, preferably SEQ ID NG's: 35 Io 38, In which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the FR sequences are disregarded; and in which b. preferably 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 2; and c. optionally said compound has a dissociation constant (K_D) to a member of the group consisting of plgR, FcRn and Vit Bl 2 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn of 10^"5 to 10^-12 moles/litre or less, and preferably IG^"7 to ϊθ^-12 moles/litre or less and more preferably 1G^~8 to 10^-12 moles/litre or less; or said compound has a rate of association (k_on-rate) to a member of the group consisting of plgR, FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit Bl 2 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding, of between 10² M^-1S^-1 to about 10⁷ M^'Y¹, preferably between 10³ M^'Y¹ and 10⁷ M^" Y¹, more preferably between 10⁴ M^" Y¹ and 10⁷ M⁴S^-1, such as between 10⁵ IVfY¹ and 10⁷ IVfY¹; or said compound has a rate of dissociation (k_Off rate) to a member of the group consisting of plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn₅ even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding, between Is^-1 and IG^"6 s^-1, preferably between 10^"2 s^-1 and 10^"6 s^-1, more preferably between 10^"3 s^-1 and 10^"6 s^-1, such as between 10^"4 s^-1 and IG^"6 s^-1; or said compound has an affinity a member of the group consisting of plgR,

FcRn and Vit B12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding, of less than 500 DM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

7. Compound according to aspects 2 to 5, wherein any of X and Y or both comprises at least one single variable domain directed against a target molecule and optionally another single variable domain directed against plgR, FcRn and Vit B 12 receptor, preferably human plgR, FcRn and Vit B12 receptor, more preferably human plgR and FcRn, even more preferably human FcRn, even more preferably to a human FcRn binding site that does not interfere with human IgG, e.g. human IgGl, and human serum albumin binding.

8. Compound according to claims 2 to 7, wherein the binding of the compounds directed against FcRn, plgR or Vit B 12 receptor is pH dependent.

9. A pharmaceutical composition for oral or nasal administration comprising a therapeutically effective amount of a compound of claims 2 to 8.

10. The composition of claim 9, additionally comprising a pharmaceutically acceptable enteric coating.

11. The composition of claims 9 to 10, wherein the compound comprises at least 2 single variable domains.

12. The composition of claim 11, wherein the single variable domains are nanobodies.

13. The composition of any of claims 8 to 12, wherein said compound is the compound of claim 7.

14. The composition of claim 13 that has agonistic properties towards the target molecule.

15. The composition of claim 13 that has antagonistic properties towards the target molecule.

16. The composition of any of claims 9 to 15 additionally comprising a permeability enhancer.

17, The composition of any of claims 9 to 16 additionally comprising a protease inhibitor.