WO2009080764A2 - Administration orale ou nasale de composés comprenant des séquences d'acides aminés - Google Patents

Administration orale ou nasale de composés comprenant des séquences d'acides aminés Download PDF

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WO2009080764A2
WO2009080764A2 PCT/EP2008/068053 EP2008068053W WO2009080764A2 WO 2009080764 A2 WO2009080764 A2 WO 2009080764A2 EP 2008068053 W EP2008068053 W EP 2008068053W WO 2009080764 A2 WO2009080764 A2 WO 2009080764A2
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fcrn
amino acid
human
binding
plgr
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PCT/EP2008/068053
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WO2009080764A3 (fr
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Hendricus Renerus Jacobus Mattheus Hoogenboom
Johannes Joseph Wilhelmus De Haard
Maria Gonzalez
Peter Vanlandschoot
Jan Terje Andersen
Gestuur Vidarsson
Edward Dolk
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Abylnx N.V.
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Publication of WO2009080764A3 publication Critical patent/WO2009080764A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • 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.
  • polypeptides e.g. single variable domains
  • 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 p ⁇ gR and FcRn, and the VMS 12 receptor, preferably plgR and FcRn 5 more preferably FcRn.
  • 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 ⁇ 2-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).
  • 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.,
  • FIG. 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 ⁇ 2-microglobulin at pH 6.0.
  • a monoclonal human ⁇ 2-microglobulin antibody (anti-hp2m mAb) and an anti polyclonal human ⁇ 2-microglobulin preparation were included as positive controls.
  • the ELISA values represent the mean of duplicates.
  • FIG. 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.
  • FIG. 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 15 Sequence alignments of mouse EpoR-Fc binding nanobodies.
  • FIG. 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.
  • FIG. 22 Mono and bivalent nanobodies binding to A- human FCRJI ELISA and B-b2- microglobulin.
  • FIG. 30 123 I 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.
  • Nanobodies in particular V HH sequences and partially humanized Nanobodies
  • 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.
  • K A association constant
  • Any K D value greater than 10 4 mol/liter (or any K A value lower than I O 4 M "s ) liters/mol is generally considered to indicate non-specific binding.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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) :
  • 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
  • the Biacore machine for example the Biacore 3000
  • 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.
  • 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).
  • 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.
  • the first amino acid sequence e.g.
  • 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).
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • 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.
  • Fc receptors axe involved in transcytosis recycling of proteins and other (biological) molecules.
  • 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)).
  • the invention provides building blocks, i.e.
  • the building block may also be the natural ligand or fragment of ligand. i.e. human Fc part.
  • 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).
  • hGH human growth hormone
  • EPO erythropoietin
  • 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.
  • K D dissociation constant
  • 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 10 2 M -1 S -1 to about 10 7 M -1 S -1 , preferably between 10 3 M -1 S -1 and 10 7 M -1 S -1 , more preferably between IG 4
  • 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 target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker.
  • the Invention further relates to compounds of formula II
  • IvT 1 S '1 such as between 10 5 M ' Y 1 and 10 7 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 "!
  • 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 humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.
  • 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.
  • 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.
  • compositions of the Invention may further comprise one or more permeation enhancer.
  • 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).
  • 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).
  • Nanobodies or dAbs 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 dry mixtures may be compacted and/or granulated and then be pulverized and/or sieved. If desired the compacted composition may be further coated.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the Compound of the invention reaches the bloodstream with a Tmax of less than 120 minutes.
  • the Compound of the invention reaches the bloodstream with a Tmax of less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes.
  • 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 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.
  • 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.
  • 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 ⁇ g/ml/minute of the Compound 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.
  • 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 ⁇ g/ml/minute Compound of the invention per ml of blood.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • a further therapeutic ingredient or pharmaceutically active compounds or principles
  • 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.
  • 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.
  • 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.
  • MM multiple myeloma disease
  • RCC renal cell carcinoma
  • BLPD B- lymphoproliferative disorder
  • 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, I
  • 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).
  • Example 1.4 Screening for faFeJRn HC binding Nanobodies
  • 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.
  • the clones were tested in an ELlSA binding assay setup, using the monoclonal phage pools obtained from the pH 5 and neutral pH selections.
  • Binding specificity was determined based on OD values compared to controls wells having received an irrelevant phage or no phage.
  • 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:
  • Nanobody candidates were diluted to 2 ⁇ g/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 ⁇ l 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).
  • HRP conjugated anti rnyc antibody monoclonal, mlgGl, Serotec
  • Example_1.8 Determination of shFcRn binding properties of selected Nanobody candidates by SPR
  • 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 ⁇ 2-microglobulin at pH 6.0. Thus, it is likely that the FcRn positive candidates interact with the folded human FcRn heavy chain.
  • 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 d ⁇ merie Nb218-H4 variants
  • HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl 5 3 mM EDTA, 0.005% surfactant P20) pH 7.4 (Biacore AB).
  • 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
  • 100 nM monomeric shFcRn 50 ⁇ l/ml at 25 0 C.
  • data were zero adjusted and the reference cell subtracted.
  • Data evaluation was performed using BIAevaluation 4.1 software (BIAcore AB).
  • 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.
  • FcRn-specific nanobodies 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).
  • 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.
  • 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.
  • the clones were tested in an ELISA binding assay setup.
  • 5 ug/ml plgR ectodomain was immobilized on Maxisorp microliter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS.
  • 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.
  • 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).
  • IgA competition efficiency of IgA competitive Nanobodies clones of the binding assay were tested in an ELISA competition assay setup.
  • 5 ug/ml plgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l Na 125 I (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.)
  • 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% argue 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.
  • 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.
  • anti-ml3-hrp is from Amersham.
  • 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.
  • 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.
  • VHH ⁇ nanobodies transcytosing from basolateral to apical could be used as a carrier to deliver therapeutics to the mucosal tissues.
  • VHH were tested on their ability to carry phages from the basolateral to the apical surface of MDCK cells using p ⁇ gR. Therefore, 10 6 TU 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.
  • CFU colony forming units
  • EpoR erythropoietin receptor
  • GHR growth hormone receptor
  • 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.
  • 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).
  • 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).
  • 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.
  • Binding specificity was determined based on OD values compared to controls having received no nanobody or an irrelevant one.
  • Figure 18 shows results of this blocking assay using a selection of clones binding to mEpoR.
  • mEpo mouse erythropoietin
  • R&D Systems Cat No 959-ME/CF Lot No EUP0207051 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.
  • 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.
  • Example 3.10 Titration of binding of monovalent and bivalent nanobodies to mEpoR
  • 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)
  • a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric "bait" construct, e.g.
  • Example 3.12 Cell assay to demonstrate agonist or antagonist activity of mEpoR
  • the mouse HCD57 erythro-leukemia cell line proliferates when Epo is added to the cells.
  • 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).
  • the phage were pre-incubated with 100 ug/ml human IgG (SIGMA 5 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.
  • 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).
  • 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.
  • Example 3.18 Screening for Nanobodies competing GH-GHR interaetioa or not competing GH-GHR
  • 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 are screened for their ability not to block GH binding to GHR/Fc.
  • 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.
  • these non-neutralizing Nanobodies are used preferably for the construction of agonistic construct comprising e.g. 2 GHR non-neutralizing Nanobodies.
  • 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).
  • 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. ⁇ soflurane 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.
  • 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 CO 2 inhalation 8 h or 4 d later and whole blood is obtained by cardiac puncture.
  • 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
  • a enterically coated capsule for mouse consumption e.g. capsule from example 32.
  • Mice are killed by CO 2 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.
  • 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.
  • 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).
  • 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).
  • enteric encapsulation of Nanobodies could also be used to protect the Nanobodies against degradation.
  • 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).
  • the body weight of the mice could be determined daily.
  • 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.
  • a similar in vivo setup to demonstrate oral to systemic delivery could be performed with Nanobodies that agonize (or antagonize) the mouse Epo receptor.
  • 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.
  • 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
  • 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.
  • human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-welI plates (Nunc 5 Wiesbaden, Germany) at 100 ⁇ g/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.
  • TEA tiiethanolamine
  • phage libraries were incubated with antigen at physiological pH and eluted at acidic pH as follows.
  • 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.
  • CPAT CPA/0.05%Tween20
  • 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.
  • Example 4.6 Selection for conditional or pH-sensitive binding of Nanobodies to albumin by ELISA.
  • 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.
  • CPAT CPA/0.05%Tween20
  • 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.
  • 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.
  • Example 4.7 Screening of Nanobody repertoire for the occurrence of a pH-sensitive interaction via surface plasmon resonance (BIAcore).
  • Nanobodie ⁇ to albumin (Biacore) are as follows: 10mM Sodium citrate (Na 3 C 6 H 5 O 7 ) + 1OmM Sodium phosphate (Na 2 HPO 4 ) + 1OmM Sodium Acetate (CH 3 COONa) + 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.
  • 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
  • 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 4 0 C with 100 ⁇ l of a 1 ⁇ g/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).
  • PBST PBS containing 0.05% Tween20
  • RT room temperature
  • PBSM room temperature
  • 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 (lOO ⁇ l) 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 ⁇ l 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 ⁇ l of a 1 : 1000 dilution of a goat anti-mouse antibody conjugated with horseradish peroxidase.
  • 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.
  • Example 5.2 Retention of conditional binding upon foriMttm ⁇ Bto . jiijiti ⁇ iedfie format
  • conditional pH- binding properties of the anti-FcRn or plgR Nanobody or dAbs within the multispecific nanobody formats 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.
  • BiAcore surface plasmon resonance
  • 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
  • Nanobodies to FcRn or plgR are as follows: 1OmM Sodium citrate (Na ⁇ C ⁇ .sO ⁇ ) + 1 OmM Sodium phosphate (Na 2 HPO 4 ) + 1OmM Sodium Acetate (CH 3 COONa) + 115mM NaCl. This mixture is brought to pH7, pH ⁇ 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.
  • 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 4 0 C with 100 ⁇ l of a 5 ⁇ g/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 ⁇ l/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.
  • the plates are washed three times (PBS containing 0.1% Tween20) and incubated for 1 hour at RT with 100 ⁇ l 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 ⁇ l 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).
  • Example 6 Preparation of various pharmaceutical orally deliverable compositions a) Capsules comprising the Compounds of the invention - preferably enteric coated capsules
  • 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),
  • 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.).
  • 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,
  • 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)
  • enteric coated capsules
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • a permeability enhancer such as e.g. acyleamitme or N ⁇ (5-chlorosalicyloyl)-8-aminocaprylic acid.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition of any of aspects 31 to 44, wherein the compound has agonistic properties to the target molecule is provided.
  • composition of any of aspects 31 to 44, wherein the compound has antagonistic properties to the target molecule is provided.
  • composition of aspect 45 wherein the compounde is an agonist or antagonist to Epo- R 5 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.
  • composition of aspect 45 wherein the compound is an agonist or antagonist to Epo-R, preferably human or mouse Epo-R.
  • composition of any of aspects 1 to 48 wherein the compound is proteolytically stabilized e.g. is selected for proteolytic stability.
  • 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.
  • 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 comprising the step of orally or nasally administering a composition according to any of aspects 1 to 50 to said subject.
  • 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.
  • 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 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.
  • 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".
  • 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 u dAb" that is directed against a target in the kidney or bladder 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.
  • a method for the prevention and/or treatment of a disease or disorder of the kidney or bladder 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.
  • 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 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.
  • a method for the prevention and/or treatment of a tumor related disease or disorder 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.
  • 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.
  • 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.
  • 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 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.
  • 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 .
  • an autoimmune disease such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease.
  • 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 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.
  • 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
  • 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).
  • TTP thrombotic thrombocytopenic purpura
  • 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).
  • TTP thrombotic thrombocytopenic purpura
  • 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 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.
  • 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.
  • MM multiple myeloma disease
  • RCC renal cell carcinoma
  • BLPD B-lymphoproliferative disorder
  • prostate cancer bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis,
  • Kaposi's sarcoma 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).
  • 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.
  • a compound, a Nanobody, a domain antibody, a single domain antibody or a "dAb” as described herein and that is directed against IL-6, ⁇ L-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.
  • MM multiple myeloma disease
  • RCC renal cell carcinoma
  • BLPD B-lymphoproliferative disorder
  • 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).
  • 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 (na ⁇ ve 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.
  • 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”.
  • 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
  • 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
  • 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.
  • 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 .
  • any of CDR ⁇ , 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 I 5 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
  • said compound has an affinity to a member of the group consisting of p ⁇ gR, 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.
  • 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).
  • 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.
  • human 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 1 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 10 7 M ' V 1 , preferably between 10 3 M -1 S -1 and IG 7 M " V ⁇ more preferably between 10 4 IvT 1 S " ' and 10 7 M 4 S -1 , such as between 10 5 M -1 S '1 and 10 7 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.
  • plgR, FcRn and Vit B 12 receptor preferably human plgR, FcRn and Vit Bl 2 receptor,
  • 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.
  • 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 ⁇ D 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
  • 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
  • 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.
  • 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%.
  • 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 10 2 M ' V 1 to about 10 7 M 4 S -1 , preferably between 10 3 M 4 S -1 and 10 7 M ' V
  • said compound has a rate of dissociation (Ic 0J p 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.
  • plgR, FcRn and Vit Bl 2 receptor preferably
  • 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).
  • a light chain variable domain sequence e.g. a V L -sequence
  • a heavy chain variable domain sequence e.g. a Vi-j-sequence
  • human IgGl and human serum albumin binding, of between 10 2 M ⁇ l s ⁇ ] to about 10 7 M ' V 1 , preferably between I O 3 M 1 V 1 and 10 7 JVT 1 S -1 , more preferably between 10 4 IVfV 1 and 10 1 IVT 1 S -1 , such as between 10 J NT's -1 and 10 7 M 4 S -1 ; or said compound has a rate of dissociation (k of f 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 5 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 iCf 6 s -1 , such as between 10 4 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.
  • 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.
  • a target molecule such as e.g. human serum albumin, human EPO-receptor or a human growth hormone, optionally linked by a linker.
  • 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
  • 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(X 6 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 ⁇ M, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • CDRl, CDR2 and CDR3 are 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 ⁇ ; ⁇ 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%.
  • said compound has a dissociation constant (K D ) to human FCR ⁇ J of 1(T 5 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 o ⁇ -rate) to human FcRn of between ] O 2 M " V ! to about 10 7 M " V ! .
  • K D dissociation constant
  • 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.
  • 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.
  • a compound as herein described e.g. a compound as described in aspects 96 to 121. for use as a medicament.
  • 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.

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Abstract

La présente invention porte sur des composés comprenant des séquences d'acides aminés tels que des domaines variables uniques pour une administration orale ou nasale et sur des compositions pharmaceutiques comprenant lesdits composés. Plus particulièrement, l'invention porte sur des composés dans lesquels la liaison à au moins l'un des antigènes ou épitopes choisis dans le groupe des transporteurs épithéliaux sert à augmenter la biodisponibilité du composé in vivo. La présente invention porte de plus sur divers procédés supplémentaires pour améliorer la biodisponibilité desdits composés de l'invention lors de l'utilisation d'une administration orale ou nasale.
PCT/EP2008/068053 2007-12-20 2008-12-19 Administration orale ou nasale de composés comprenant des séquences d'acides aminés WO2009080764A2 (fr)

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EP4007591A4 (fr) * 2019-08-02 2023-11-29 Janssen Biotech, Inc. Matières et procédés pour le biotransport multidirectionnel
US11926669B2 (en) 2022-05-30 2024-03-12 Hanall Biopharma Co., Ltd. Anti-FcRn antibody or antigen binding fragment thereof with improved stability
WO2024107885A1 (fr) * 2022-11-15 2024-05-23 Vanderbilt University Conjugués nanocorps-médicament et procédés de préparation associés

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US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US20110311515A1 (en) * 2009-01-14 2011-12-22 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
AU2010205627B2 (en) * 2009-01-14 2015-04-02 Ablynx Nv Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US8999669B2 (en) 2009-07-06 2015-04-07 Ludwig-Maximilians Universitat München Detection and visualization of the cell cycle in living cells
EP2275442A1 (fr) * 2009-07-06 2011-01-19 Ludwig-Maximilians-Universität München Détection et visualisation du cycle cellulaire dans des cellules vivantes
WO2011003896A1 (fr) * 2009-07-06 2011-01-13 Ludwig-Maximilians-Universität Détection et visualisation du cycle cellulaire dans des cellules vivantes
US10233243B2 (en) 2012-05-14 2019-03-19 Ucb Biopharma Sprl Anti-FcRn antibodies
CN104364265B (zh) * 2012-05-14 2019-07-16 Ucb生物制药私人有限公司 抗FcRn抗体
CN104364265A (zh) * 2012-05-14 2015-02-18 Ucb医药有限公司 抗FcRn抗体
US11384148B2 (en) 2012-05-14 2022-07-12 UCB Biopharma SRL Anti-FcRn antibodies
EP3527588A1 (fr) * 2012-05-14 2019-08-21 UCB Biopharma SPRL Anticorps anti-fcrn
WO2014019727A1 (fr) * 2012-05-14 2014-02-06 Ucb Pharma S.A. Anticorps anti-fcrn
EA032770B1 (ru) * 2012-05-14 2019-07-31 Юсб Фарма С.А. АНТИТЕЛА ПРОТИВ FcRn
US11220547B2 (en) 2013-11-12 2022-01-11 Ucb Biopharma Sprl Antibodies specific to FCRN
CN105814080B (zh) * 2013-11-13 2019-09-24 Ucb生物制药私人有限公司 特异于fcrn的抗体
US10273302B2 (en) 2013-11-13 2019-04-30 Ucb Biopharma Sprl Antibodies specific to FcRn
WO2015071330A1 (fr) * 2013-11-13 2015-05-21 Ucb Biopharma Sprl Anticorps spécifiques à fcrn
EP3572433A1 (fr) * 2013-11-13 2019-11-27 UCB Biopharma SPRL Anticorps spécifiques à fcrn
EA035142B1 (ru) * 2013-11-13 2020-05-06 Юсб Биофарма Спрл Антитела, специфичные к fcrn
CN105814080A (zh) * 2013-11-13 2016-07-27 Ucb生物制药私人有限公司 特异于fcrn的抗体
KR102366114B1 (ko) 2013-11-13 2022-02-21 유씨비 바이오파마 에스알엘 FcRn에 특이적인 항체
KR20160077211A (ko) * 2013-11-13 2016-07-01 유씨비 바이오파마 에스피알엘 FcRn에 특이적인 항체
WO2016128974A1 (fr) * 2015-02-09 2016-08-18 Entera Bio Ltd. Formulations pour administration orale d'agents actifs
US10583177B2 (en) 2015-02-09 2020-03-10 Entera Bio Ltd. Formulations for oral administration of active agents
EP4007591A4 (fr) * 2019-08-02 2023-11-29 Janssen Biotech, Inc. Matières et procédés pour le biotransport multidirectionnel
US11926669B2 (en) 2022-05-30 2024-03-12 Hanall Biopharma Co., Ltd. Anti-FcRn antibody or antigen binding fragment thereof with improved stability
WO2024107885A1 (fr) * 2022-11-15 2024-05-23 Vanderbilt University Conjugués nanocorps-médicament et procédés de préparation associés

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