Classifications

• • 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
• • 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
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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®

Definitions

• the present invention relates to the oral delivery of polypeptides. More particularly, the present invention relates to the oral delivery of polypeptides comprising a single variable domain such as a Nanobody®, a domain antibody, a single domain antibody, a "dAb” or formatted version thereof, e.g. Polypeptides comprising Nanobodies having multivalent or multimeric binding properties (herein Polypeptides of the Invention, see also further description herein for a more detailed description).
• the present invention provides compositions suitable for oral delivery of said Polypeptides of the Invention.
• the invention also relates to methods for the treatment of a subject comprising the delivery of said
• Polypeptides of the Invention to said subject by the oral route and to methods-of enhancing bioavailability of such Polypeptides of the Invention when administered orally.
• Therapeutic peptides and proteins are often unstable, have large molecular weights and are polar in nature. These properties lead to poor permeability through biological membranes. When administered orally, they are susceptible to proteolytic degradation in the gastrointestinal tracts and only pass with difficulty into the body fluids. For this reason, therapeutic peptides and proteins have hitherto been administered mostly by injection, infusion or oral delivery.
• Proteolytic enzymes of both the stomach and intestines may degrade polypeptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of polypeptides that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas (typically having neutral to basic pH optima). Specific difficulties arising from the oral administration of a polypeptide involve the relatively large size of the molecule, and the charge distribution it carries. This may make it more difficult for a polypeptide to penetrate the mucus along intestinal walls or to cross the intestinal brush border membrane into the blood.
• Oral administration of polypeptides has 2 main challenges that are a) degradation by proteolytic enzymes in the stomach and intestine and b) poor absorption, i.e. poor transport of said polypeptide from the apical to the baso lateral side of the intestine and release into the blood. Improving oral effectiveness, i.e. increase of the bioavailability of oral polypeptidic drugs, is a clear unmet medical need and important for several reasons.
• peptides and proteins are expensive to manufacture either by chemical synthesis or recombinant DNA technologies. Therefore, the more one increases bioavailability, the lesser the amounts that will be required in an oral formulation of a therapeutic drug (economic issue).
• polypeptides of the Invention i.e. the Polypeptides of the Invention (and further described herein below), generally also including peptides but preferably polypeptides that are larger than 100 amino acids in length, can be delivered into the bloodstream via the oral route.
• the Polypeptides of the Invention can be conveniently administered to a subject by the oral route by means of a composition comprising said Polypeptides of the Invention with the relevant strategies as disclosed herein.
• Said Polypeptides of the Invention are characterized and partly shown to be one of the following a) more protease resistant than conventional biologies, e.g.
• conventional antibodies b) have typically a higher pH stability or as shown herein (can bind in a pH dependent manner), c) have typically a high temperature stability (i.e. having advantages during processes requiring high T, i.e. in processes of formulation, i.e. compaction and/or granulation), d) have typically a high stability to organic solvents, i.e. may show a superior stability profile to e.g. PLGA solvent exposure (PLGA or poly(lactic-co-glycolic acid) is an Food and Drug Administration (FDA) approved copolymer which is used in a host of therapeutic devices), e) have shown to have long time stability, f) are typically small globular domains (e.g. in a monovalent form are about 10 times smaller than conventional antibodies) allowing for high loading capacity of matrix or implant, and/or g) have typically high solubility allowing for high loading and highly concentrated doses.
• PLGA solvent exposure PLGA or poly(lactic-co-glycolic acid)
• the invention provides one or more of the following main strategies to achieve orally administered polypeptide delivery: a) inhibit proteolytic activity that degrades polypeptides in stomach and gut, b) develop protease-resistant polypeptide analogs that retain biological activity, c) stabilize the polypeptide by conjugation to shielding molecules, d) protect the polypeptide from proteolytic degradation by e.g. enteric coating, e) improve passive polypeptide transport (diffusion) through the epithelial membrane of the intestine, f) improve active (e.g. receptor mediated or M-cell mediated) trans-epithelial transport of the polypeptides, and/or g) increase half-life of the polypeptide in human body, e.g.
• 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.
• suitable excipient e.g. biodegradable polymer
• the Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers).
• the Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g.
• plgR, FcRn, and/or VitBl 2 receptor mediated trans-epithelial transport preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• suitable excipient e.g. biodegradable polymer
• the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
• active e.g. receptor mediated
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); 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.
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies. systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); 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.
• active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• suitable excipient e.g. biodegradable polymer
• covalently binding an unit allowing for longer half life e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans- epithelial transport in the gut.
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); 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.
• permeation enhancer such as acylcarnitine and/or Eligeii® carrier technology
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); 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.
• enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers)
• 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.
• nM preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH ⁇ or less but has 2 times less, preferably 3, 4, 5. 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent plgR, pH dependent FcRn 5 and/or pH dependent VitBl 2 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.
• active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder; and [d) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or e) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcarnitine and/or Eligen® carrier technology].
• suitable excipient e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the ait, e.g. Eudragit L3OD-55 (Roehm Pharma Polymers); and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by plgR, FcRn, and/or VitB12 receptor mediated trans -epithelial transport, preferably plgR and/or FcRn.
• active e.g. receptor mediated
• FcRn mediated trans-epithelial transport More preferably FcRn mediated trans-epithelial transport; and [c) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or d) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcarnitine and/or Eligen® carrier technology].
• protease inhibitors such as e.g. organic acids
• permeation enhancer such as acylcarnitine and/or Eligen® carrier technology
• polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); and b) providing continuous local (topical in gut) delivery by bacterial system, e.g. lactit acid bacteria.
• the present invention accordingly, relates to a method for the delivery or administration (both terms are used interchangeably throughout the invention) of a Polypeptide of the Invention to the bloodstream and/or other organ and/or tissue (e.g. the kidney, heart, liver, bladder, lung and/or brain) of a subject without being substantially inactivated (i.e. maintaining to a large part its functionality or delivery is such that a safe and efficacious delivery to the target side is provided), comprising the step of administering to said subject by the oral route a composition comprising said Polypeptide of the Invention.
• organ and/or tissue e.g. the kidney, heart, liver, bladder, lung and/or brain
• the present invention provides a pharmaceutical composition (hereafter referred to as the Pharmaceutical Composition of the Invention) comprising the Polypeptide of the Invention, wherein said polypeptide is designed at least partly in such a way as disclosed herein.
• the Polypeptide of the Invention has an amino acid sequence that at least comprises one or more single variable domain(s), e.g. a Nanobody, a domain antibody, a single domain antibody or a '"dAb".
• the Polypeptide of the Invention has an amino acid sequence essentially consisting of one or more single variable domain(s). e.g. a Nanobody, a domain antibody, a single domain antibody or a "dAb”.
• the Polypeptide of the Invention has an amino acid sequence essentially consisting of one or more single variable domain(s), e.g. a Nanobody (which is also called the Nanobody of the Invention).
• a Nanobody which is also called the Nanobody of the Invention.
• the Nanobody, domain antibody, single domain antibody or "dAb' * is derived from a V H or V HH - AS described further in the detailed description
• the Polypeptide of the Invention comprises a single amino acid chain that can be considered to comprise "framework sequences" or "PR's" and "complementarity determining regions" or "CDR' s”.
• amino acid sequences of the Nanobodies and/or Polypeptides of the Invention can be "humanized”, “camelized” or modified as further described herein.
• Polypeptides of the Invention that comprise or essentially consist of a single variable domain, e.g. a single Nanobody, domain antibody, single domain antibody or "dAb” will be referred to herein also as “monovalent” polypeptides or as “monovalent constructs " '.
• Polypeptides of the Invention that comprise or essentially consist of two or more single variable domains e.g. Nanobodies, domain antibodies, single domain antibodies or u dAb's” will be referred to herein also as “multivalent” polypeptides or as “multivalent constructs", and these may provide certain advantages compared to the corresponding monovalent single variable domains, e.g. Nanobodies, domain antibodies, single domain antibodies or "dAb's”.
• Polypeptides of the Invention comprise or essentially consist of at least two single variable domains, e.g. Nanobodies, domain antibodies, single domain antibodies or "dAb's", such as two or three, preferably two, single variable domains, e.g. Nanobodies, domain antibodies, single domain antibodies or "dAb's".
• such multivalent constructs can provide certain advantages compared to a polypeptide comprising or essentially consisting of a single variable domain, such as a single Nanobody, domain antibody, single domain antibody or "dAb”, such as a much improved affinity and/or specificity for its antigen. It will be clear for the skilled person how to make such a multivalent constructs from the disclosure herein.
• Polypeptides of the Invention comprise or essentially consist of at least one Nanobody, domain antibody, single domain antibody or "dAb” directed 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 of the same target, antigen, target, protein or polypeptide.
• Such polypeptides are also referred to herein as "multispecific” polypeptides or as 'multispecific constructs", and these may provide certain advantages compared to the corresponding monovalent or monospecific Nanobodies, domain antibodies, single domain antibodies or “dAb's". It will be clear for the skilled person how to make such multispecific constructs from the disclosure herein.
• Polypeptides of the Invention comprise or essentially consist of at least one Nanobody, domain antibody, single domain antibody or "dAb", optionally one or more further Nanobodies, domain antibodies, single domain antibodies or “dAb's” and at least one other amino acid sequence that adds at least one desired property to the Nanobody, domain antibody, single domain antibody or "dAb” and/or to a resulting fusion protein.
• fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies, domain antibodies, single domain antibodies or "dAbs”.
• said at least one other amino acid sequence provides an increased half-life to the Polypeptides of the Invention without said other amino acid sequence.
• said at least one other amino acid sequence e.g. Fc polypeptide, allows the Polypeptides of the Invention to be directed towards, penetrate and/or cross the mucosal membrane and/or the blood brain barrier,
• the one or more Nanobodies, domain antibodies, single domain antibodies or “dAbs” and/or other amino acid sequences may be directly linked or linked via one or more linker sequences.
• linkers may also be amino acid sequences, e.g. Ala-Ala-Ala, Gly-Gly-Gly (3-Gly), 9-Gly, or 30-Gly sequence, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).
• Polypeptides of the Invention either comprise one or more Nanobodies, domain antibodies, single domain antibodies or "dAb's", optionally linked via one or two linkers, or is a multispecific polypeptide, comprising one or more Nanobodies, domain antibodies, single domain antibodies or "dAb's” and at least one Nanobody, domain antibody, single domain antibody or "dAb' * that provides an increased half-life following delivery to the subject, particularly providing extended metabolic persistence in an active state within the physiological environment (e.g., in the stomach, at the mucosal surface, in the bloodstream, and/or within another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain).
• the physiological environment e.g., in the stomach, at the mucosal surface, in the bloodstream, and/or within another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain.
• Nanobody, domain antibody, single domain antibody or "dAb” directed against a FcRn, and in particular human FcRn, serum protein, and in particular against a human serum protein, such as against human serum albumin, in which said Nanobodies, domain antibodies, single domain antibodies or “dAb's” again optionally linked via one or more linkers. It will be clear for the skilled person how to make such constructs from the disclosure herein.
• a Polypeptide of the Invention comprises one or more (such as two or preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb V linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting
• said one or more amino acid sequences that allow the resulting Polypeptides of the Invention to cross the intestine wall may be one or more Nanobodies, domain antibodies, single domain antibodies or "dAb's” directed against an M-cell-specific molecule on the epithelial membrane, wherein said Nanobodies, domain antibodies, single domain antibodies or "dAb's” cross the mucosal membrane upon binding to said epithelial transmembrane protein.
• Mucosa-associated lymphoid tissue in the digestive tracts are covered by a specialized epithelium, the follicle- associated epithelium, which includes M cells, which are specialized for the uptake and transcytosis of macromo ⁇ ecules and microorganisms. Following transcytosis, antigens are released to cells of the immune system in lymphoid aggregates beneath the epithelium where antigen processing and presentation and stimulation of specific B and T lymphocytes are achieved. Circulation of the lymphoid cells enables their homing to their original, and other, mucosal sites where they exert the effector function. Such a response may be dominated by secretory immunoglobulin A release and may include cytotoxic T lymphocyte action.
• Binding of particles to the apical M cell membrane may be nonspecific or due to specific interaction between molecules such as integrins and lectins. Exploiting the specific binding to M cells is an aim for example to increase the efficiency of uptake of an orally delivered polypeptide by its conjugation to an M-cell-specific molecule.
• said one or more amino acid sequences that allow the resulting Polypeptides of the Invention to cross the intestine wall may be one or more Nanobodies, domain antibodies, single domain antibodies or "dAb's" directed against the human polymeric immunoglobulin receptor, hpIgR, and/or FcRn, in particular human FcRn.
• a Polypeptide of the Invention comprises one or more (such as two or preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb's" linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that confer an increased half-life in vivo to the resulting Polypeptide of the Invention, in particular, that provides extended metabolic persistence in an active state within the physiological environment (e.g. at the gut epithelial surface, in the bloodstream and/or within another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain).
• the physiological environment e.g. at the gut epithelial surface, in the bloodstream and/or within another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain.
• said amino acid sequences that confer an increased half-life in vivo to the resulting Polypeptide of the Invention may be one or more (such as two and preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb's", and in particular Nanobodies, domain antibodies, single domain antibodies or "dAb's” directed against a human serum protein such as human serum albumin.
• suitable Nanobodies against mouse or human serum albumin are described in the applications WO 03/035694, WO 04/041865 and WO 06/122825.
• a polypeptide or protein of the invention comprises one or more (such as two or preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb's", one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to be directed towards, penetrate and/or cross the mucosal membrane, and one or more (such as two and preferably one) amino acid sequences that confer an increased half-life in vivo to the resulting polypeptide of the invention, in particular, that provides extended metabolic persistence in an active state within the physiological environment (e.g. at the gut mucosal surface, in the bloodstream and/or within another selected physiological compartment, tissue and/or organ such as e.g.
• said one or more amino acid sequences that allow the resulting polypeptides of the invention to be directed towards, penetrate and/or cross the mucosal membrane or to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb's" (as mentioned herein), and said amino acid sequences that confer an increased half-life in vivo to the resulting polypeptide of the invention may be one or more (such as two and preferably one) Nanobodies, domain antibodies, single domain antibodies or "dAb's” (also as mentioned herein).
• compositions of the present invention are formulated for oral administration. Accordingly, in addition to the Polypeptides of the invention, e.g. constructs comprising single variable domains such as e.g. Nanobodies binding to a target molecule and to e.g. FcRn, plgR or/and VitB12 receptors, the composition of the invention may also comprise a pharmaceutically acceptable oral carrier and, optionally, other therapeutic ingredients or pharmaceutically acceptable additives and/or agents.
• a pharmaceutically acceptable oral carrier and, optionally, other therapeutic ingredients or pharmaceutically acceptable additives and/or agents.
• the present invention relates to a composition that comprises at least a Polypeptide of the Invention (e.g. humanized and e.g. formatted with human FcRn binding unit), optionally an enteric coating (in order to protect said polypeptide from proteolytic), preferably an enteric coating, and at least one further excipient selected from the group consisting of: a) protease inhibitor such as an organic acid); b) proton pump inhibitor such as omeprazole or any other -zoles; c) tonicifiers, d) osmolytes, and/or without being limiting e) surfactants.
• a) protease inhibitor such as an organic acid
• proton pump inhibitor such as omeprazole or any other -zoles
• tonicifiers such as omeprazole or any other -zoles
• surfactants e.g. Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy) 21 st edition.
• composition of the invention also comprises other additives and/or agents.
• a composition comprising a Polypeptide of the Invention (e.g. humanized and e.g. formatted with human FcRn binding unit), optionally an enteric coating (in order to protect said polypeptide from proteolytic), preferably an enteric coating, and one or more pharmaceutically acceptable additives and/or agents.
• a Polypeptide of the Invention e.g. humanized and e.g. formatted with human FcRn binding unit
• enteric coating in order to protect said polypeptide from proteolytic
• enteric coating preferably an enteric coating
• the composition of the invention may comprise a Polypeptide of the Invention (e.g. humanized and e.g. formatted with human FcRn binding unit), optionally an enteric coating (in order to protect said polypeptide from proteolytic), preferably an enteric coating, and optionally, one or more additives and/or agents.
• a Polypeptide of the Invention e.g. humanized and e.g. formatted with human FcRn binding unit
• an enteric coating in order to protect said polypeptide from proteolytic
• an enteric coating preferably an enteric coating
• additives and/or agents optionally, one or more additives and/or agents.
• the composition may additionally comprise one or more further therapeutic ingredient (or active substances).
• further therapeutic ingredient or active substances.
• the present invention also provides methods for the preparation of a Composition of the Invention. Those methods will also become clear from the further description herein.
• compositions of the invention are capable of providing a systemic therapeutic or biological activity of the Polypeptide of the Invention, preferably a Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, in a subject, following oral administration of said composition comprising said Polypeptide of the Invention to said subject.
• the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody reaches a Cmax in blood of at least 1 ng of Polypeptide comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, per ml of blood.
• the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, per ml of blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, reaches the bloodstream with a Tmax of less than 120 minutes.
• the Polypeptide comprising at least a Nanobody and/or dAbs, more preferably a Nanobody reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention comprising at least a Nanobody and/or dAbs. more preferably a Nanobody, per ml of blood within less than 120 minutes following oral administration of the composition comprising said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody reaches a Cmax in blood of at least 1 ng of Polypeptide of the
• Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, per ml of blood within less than 120 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the AUC for the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, in blood following oral administration of a composition comprising said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody is at least 500 ng/ml/minute Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody
• the AUC for the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, in blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody is at least 500 ng/ml/minute Polypeptide comprising at least a Nanobody and/or dAbs, more preferably a
• the bioavailability for the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, in blood following oral administration of a composition comprising said Polypeptide comprising at least a Nanobody and/or dAbs, more preferably a Nanobody is at least 1%, preferably 2%, 3% or 4%, more preferably 5%, most preferred 10%, compared to parenteral administration of said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the Composition of the Invention is capable of providing a therapeutic or biological activity of the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, in the blood of a subject, following oral administration to said subject of a composition comprising said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the bioavailability for the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs.
• Nanobody in the blood following oral administration of a composition comprising said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody is at least 1%, preferably 2%, 3% or 4%, more preferably 5%, most preferred 10%, compared to parenteral administration of said Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody.
• the invention further provides a method for delivering a Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, to the bloodstream of a subject without being significantly inactivated or to only such an extent to still fulfill its biological function, said method comprising the step of orally administering a Composition comprising a Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, to said subject.
• the present invention also provides methods for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a Nanobody, comprising the step of orally administering to said subject a composition as described above and/or below. Further therapeutic applications of the compositions of the invention are described in detail hereafter.
• Figure 1 hFcRn HC binding assay at different pH for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition. DETAILED DESCRIPTION
• Target Molecule or “Target Molecules” or “target” is meant a protein with a biological function in an organism, preferably animal, more preferably mammal most preferred human, wherein said biological function may be involved in the initiation or progression or maintenance of a disease.
• said protein is selected from the group consisting of: human growth hormone(hGH), N- methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B- chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormones(FSH), thyroid stimulating hormone(TSH), and leutinizing hormone(LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, a Nanobody, a molecule which is well tolerated by mammals in particularly humans and has a long half life when given systemically and/or locally, e.g. poly glycol chains of different size, e.g.
• PEG-20, PEG-30 or PEG40 lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkepha ⁇ inase, human serum albumin, mullerian- inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, such as betalactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, receptors for hormones or growth factors; integrin, thrombopoietin, protein A or D, rheumatoid factors, nerve growth factors such as NGF- ⁇ , platelet-growth factor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD
• a multimeric protein is a protein which is associated (typically by non-covalent interactions) in biological organism such as humans with others as subunits in a multimeric structure and typically only in the multimeric format is able to unfold its biological function. This is also called the quaternary structure of the protein. This association can also be stabilized by disulfide bonds and by noncovalent interactions with reacting substrates or cofactors.
• the single variable domains that are present in the constructs of the invention may be any variable domain that forms a single antigen binding unit.
• such single variable domains will be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR' s, as further described herein).
• Such single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.
• the single variable domain may for example comprise a light chain variable domain sequence (e.g. a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g.
• V H -sequence or V HH sequence or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and ScFv fragments that need to interact with another variable domain - e.g. through a V H AV 1 interaction - to form a functional antigen binding domain).
• a single antigen binding unit i.e. a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and ScFv fragments that need to interact with another variable domain - e.g. through a V H AV 1
• the single variable domain may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) or a NanobodyTM (as defined herein, and including but not limited to a V HH sequence); other single variable domains, or any suitable fragment of any one thereof.
• (single) domain antibodies reference is also made to the prior art cited above, as well as to EP 0 368 684.
• the amino acid sequence of the invention may be a NanobodyTM or a suitable fragment thereof.
• NanobodyTM, NanobodiesTM and NanocloneTM are trademarks of Ab lynx N. V.J
• V HH ' S and Nanobodies reference is made to the review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and
• WO 96/34103 of the Vrije Universiteit Brussel WO 94/25591, WO 99/3768I 5 WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N. V. and Ablynx N.V.; WO
• Nanobodies in particular V HH sequences and partially humanized Nanobodies
• V HH sequences and partially humanized Nanobodies can in particular be characterized by the presence of one or more "Hallmark residues ' " in one or more of the framework sequences.
• a further description of the Nanobodies. including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non- limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO07/104529.
• high affinity as used herein is meant a dissociation constant for a monovalent binding Nanobody of (Kd) of ⁇ 100 nM and preferably 10 nM and more preferably InM and even more preferably lOOpM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art.
• high avidity as used herein is meant a dissociation constant for a bi- or multivalent binding Nanobody of (Kd) of ⁇ 100 nM and preferably 10 nM and more preferably InM and even more preferably 10OpM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art.
• rigid secondary structure any polypeptide segment exhibiting a regular repeated structure such as is found in; ⁇ -helices, 310 helices, ⁇ - helices, parallel and antiparallel ⁇ -sheets, and reverse turns. Certain "non-ordered" structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or "patch" of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns.
• the geometry of these rigid secondary structures is well defined by ⁇ and .psi.
• the requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or "patch" of amino acid residues that can be exposed to and bind with a target molecule. It is primarily these amino acid residues that are replaced by mutagenesis that form the "library” of structurally related (mutant) binding polypeptides that are displayed on the surface of the phage and from which novel polypeptide ligands are selected. Mutagenesis or replacement of amino acid residues directed toward the interior of the polypeptide is generally avoided so that the overall structure of the rigid secondary structure is preserved.
• leader sequence as used herein is meant a particular section of messenger RNA (mRNA) and the DNA that codes for it. It starts at the +1 position (where transcription begins) and ends just before the start codon (usually AUG) of the coding region. It usually contains a ribosome binding site (RBS), in bacteria also known as the Shine-Delgarno sequence (AGGAGGU).
• RBS ribosome binding site
• the 5 1 UTR may be a hundred or more nucleotides long, and the 3' UTR may be even longer (up to several kilobases in length) (Molecular Cell Biology, 5th edition, Lodish et al. pi 13, chapter 4.2). Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual” ( 2nd.Ed.), VoIs. 1-3, Cold Spring Harbor Laboratory Press (1989); F.
• immunoglobulin sequence whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as V HH domains or V H /V L domains, respectively).
• sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “V H H sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
• nucleotide sequence as used herein also encompasses a nucleic acid molecule with said nucleotide sequence, so that the terms “nucleotide sequence” and “nucleic acid” should be considered equivalent and are used interchangeably herein; h) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640- 56; Levin and Weiss, MoI.
• the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.
• a known computer algorithm for sequence alignment such as NCBI Blast v2.0
• Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768- ⁇ .
• the nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence;
• the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence ⁇ and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence -
• amino acid difference as defined herein.
• degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
• amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.
• amino acid substitutions can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide.
• Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.
• Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) ⁇ (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar 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, nonpolar residues: Met, Leu, lie, VaI and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
• Particularly preferred conservative substitutions are as follows: Ala into GIy or into Ser; Arg into Lys; Asn into GIn or into His; Asp into GIu; Cys into Ser; GIn into
• Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer- Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et ai., Proc. Nad. Acad Sci. USA 81 : 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 198 I 5 and Goldman et al., Ann.
• amino acid sequences and nucleic acid sequences are said to be “exactly the same' ' ' if they have 100% sequence identity (as defined herein) over their entire length; m) When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences; n) When a nucleotide sequence or amino acid sequence is said to "comprise” another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the firstmentioned nucleotide
• a Nanobody of the invention when a Nanobody of the invention is said to comprise a CDR sequence, this may mean that said CDR sequence has been incorporated into the Nanobody of the invention, but more usually this generally means that the Nanobody of the invention contains within its sequence a stretch of amino acid residues with the same amino acid sequence as said CDR sequence, irrespective of how said Nanobody of the invention has been generated or obtained.
• the latter amino acid sequence when it has a specific biological or structural function, it preferably has essentially the same, a similar or an equivalent biological or structural function in the firstmentioned amino acid sequence (in other words, the firstmentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, a similar or an equivalent biological or structural function).
• the CDR sequence and framework are preferably capable, in said Nanobody, of functioning as a CDR sequence or framework sequence, respectively.
• nucleotide sequence when a nucleotide sequence is said to comprise another nucleotide sequence, the ftrstmentioned nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the firstmentioned, larger nucleotide sequence).
• an expression product e.g. a polypeptide
• a nucleic acid sequence or amino acid sequence is considered to be "(in) essentially isolated (formy - for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component.
• a nucleic acid sequence or amino acid sequence is considered “essentially isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100- fold, and up to 1000-fold or more.
• a nucleic acid sequence or amino acid sequence that is "in essentially isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylarm ' de-gel electrophoresis; p)
• domain generally refers to a globular region of an amino acid sequence (such as an antibody chain, and in particular to a globular region of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region.
• a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds.
• binding domain' ' refers to such a domain thai is directed against an antigenic determinant (as defined herein); q)
• antigenic determinant refers to the epitope on the antigen recognized by the antigen-binding molecule (such as a Nanobody or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule.
• antigenic determinant ' and epipe may also be used interchangeably herein.
• An amino acid sequence (such as a Nanobody, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against” or “directed against” said antigenic determinant, epitope, antigen or protein, s)
• the term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen- binding protein (such as a Nanobody or a polypeptide of the invention) molecule can bind.
• the specificity of an antigen-binding protein can be determined based on affinity and/or avidity.
• the affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (Ko). is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K D , the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K A ), which is 1/K D ).
• affinity can be determined in a manner known per se, depending on the specific antigen of interest.
• Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.
• antigen-binding proteins such as the amino acid sequences, Nanobodies and/or polypeptides of the invention
• Ko dissociation constant
• IO 5 to 10 i2 liter/ moles or more and preferably 10 7 to 10 12 liter/moles or more and more preferably 10 8 to 10 12 liter/moles.
• any K D value greater than 10 4 mol/iiter (or any K A value lower than 10 M " ) 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.
• Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RlA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
• the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein.
• dissociation constants may not be possible to measure dissociation constants of more then 10 " moles/liter or 10 "3 moles/liter (e,g, of 10 "2 moles/liter).
• the (actual or apparent) dissociation constant may be calculated on the basis of the
• the affinity denotes the strength or stability of a molecular interaction.
• the affinity is commonly given as by the K D , or dissociation constant, which has units of mol/liter (or M).
• the affinity can also be expressed as an association constant, K A , which equals 1/K D and has units of (mol/liter) "1 (or M '1 ).
• K D dissociation constant
• K A association constant
• the stability of the interaction between two molecules will mainly be expressed in terms of the K D value of their interaction; it being clear to the skilled person that in view of the relation K A -1/K D , specifying the strength of molecular interaction by its K D value can also be used to calculate the corresponding K A value.
• the K D for biological interactions which are considered meaningful are typically in the range of 10 "10 M (0.1 nM) to 10 "5 M (10000 nM). The stronger an interaction is, the lower is its K D .
• the off-rate k Off has units s "1 (where s is the SI unit notation of second).
• the on-rate k on has units M " V 1 .
• the on- rate may vary between 10 2 M ' V 1 to about 10 7 M -1 S "1 , approaching the diffusion- limited association rate constant for bimolecular interactions.
• the affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et ah, Intern.
• SPR surface plasmon resonance
• 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,
• a and B 5 one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated
• a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated
• the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B.
• an IC 50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved.
• K D ref the K D of the reference molecule
• the apparent K D for the interaction A-B can be obtained from following formula: K D K D re f)- Note that if c re f ⁇ c K D re f, K D ⁇ IC 50 .
• the measurement of the IC 5 0 is performed in a consistent way (e.g.
• the half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.
• the in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis.
• Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering to a warm-blooded animal (i.e. to a human or to another suitable mammal, such as a mouse, rabbit, rat, pig, dog or a primate, for example monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys ⁇ Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus)) a suitable dose of the amino acid sequence, compound or polypeptide of the invention; collecting blood samples or other samples from said animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence, compound or polypeptide of the invention has been reduced by 50% compared to the initial level upon dosing.
• an "'increase in half-life” refers to an increase in any one of these parameters, such as any two of these parameters, or essentially all three these parameters.
• “increase in half-life” or “increased half- life” in particular refers to an increase in the tl/2-beta, either with or without an increase in the tl/2-alpha and/or the AUC or both.
• “modulating” or “to modulate * ' generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of. a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay.
• modulating or ''to modulate may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least i%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.
• moduleating may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands. binding partners, partners for association into a homomultimeric or heterom ⁇ ltimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention.
• this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved.
• Modulating may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), iigand(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
• 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
• 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
• an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%. for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.
• Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target or antigen.
• Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions).
• the term "interaction site" on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an 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 involved in a biological action or mechanism of the target or antigen. More generally, an interaction site on the target or antigen means a site, epitope, antigenic determinant, part,
• interaction site can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein), w)
• An amino acid sequence or polypeptide is said to be * ' 'specific for" a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a K D value, K A value, K off rate and/or K 0n rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide.
• the first antigen may bind to the target or antigen with a K D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the K D with which said amino acid sequence or polypeptide binds to the second target or polypeptide.
• an amino acid sequence or polypeptide when an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen,
• cross-block when an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen.
• cross-block cross-blocked
• cross-blocking are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target.
• the extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to [target], and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays.
• One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology.
• Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target.
• the following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross- blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequence or other binding agents described herein.
• the Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations.
• 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.
• 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).
• Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture.
• the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence.
• the concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip.
• the amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromoiar (on a binding site basis).
• Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix.
• the test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded.
• the chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds.
• the solution of A* alone is then passed over the target-coated surface and the amount of binding recorded.
• the chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target.
• the solution of B* alone is then passed over the target-coated surface and the amount of binding recorded.
• the maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the target surface alone.
• a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination.
• the Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version (R & D Systems, Minneapolis. MN, USA; 2005 cat# 1406-ST-025).
• an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence.
• the cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface.
• C-terminal His-tagged target could alternatively be used.
• various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti- FLAG antibodies; biotin tag with streptavidin).
• the general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules.
• the plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target.
• the amount of bound target is then measured using a reagent that is appropriate to detect the target.
• An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence.
• the first amino acid sequence e.g.
• an Ab-X is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added.
• An excess amount of the second amino acid sequence, i.e. Ab-Y is then added to the ELISA plate such that the moles of Ab-Y [target] binding sites per well are at least 10 fold higher than the moles of Ab-X [target] binding sites that were used, per well, during the coating of the ELISA plate.
• [target] is then added such that the moles of [target] added per well are at least 25- fold lower than the moles of Ab-X [target] binding sites that were used for coating each well.
• the background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), [target] buffer only (i.e. no target) and target detection reagents.
• the positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X) 5 second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), target and target detection reagents.
• the ELISA assay may be ran in such a manner so as to have the positive control signal be at least 6 times the background signal.
• the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution.
• Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal ⁇ i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells).
• the total number of amino acid residues in a Nanobody can be in the region of 110-120, is preferably 112-1 15, and is most preferably 113.
• parts, fragments, analogs or derivatives (as further described herein) of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; z)
• the amino acid residues of a Nanobody are numbered according to the general numbering for V H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD.
• FRl of a Nanobody comprises the amino acid residues at positions 1-30
• CDRl of a Nanobody comprises the amino acid residues at positions 31-35
• FR2 of a Nanobody comprises the amino acids at positions 36-49
• CDR2 of a Nanobody comprises the amino acid residues at positions 50-65
• FR3 of a Nanobody comprises the amino acid residues at positions 66-94
• CDR3 of a Nanobody comprises the amino acid residues at positions 95-102
• FR4 of a Nanobody comprises the amino acid residues at positions 103-1 13.
• 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.
• position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa
• position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa
• position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.
• Nanobodies, (single) domain antibodies or “dAb's” can be derived from the variable region of a 4-chain antibody as well as from the variable region of a heavy chain antibody.
• the variable domains present in naturally occurring heavy chain antibodies will also be referred to as "V HH domains ' ", in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "F# domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "F L domains").
• the polypeptide or protein of the invention has an amino acid sequence that comprises or essentially consists of four framework regions (FRl to FR4, respectively) and three complementarity determining regions (CDRl to CDR3, respectively).
• Such an amino acid sequence preferably contains between 80 and 200 amino acid residues, such as between 90 and 150 amino acid residues, such as about 100-130 amino acid residues (although suitable fragments of such an amino acid sequence - i.e. essentially as described herein for the Nanobodies of the invention or equivalent thereto - may also be used), and is preferably such that it forms an immunoglobulin fold or such that, under suitable conditions, it is capable of forming an immunoglobulin fold (i.e. by suitable folding).
• the amino acid sequence is preferably chosen from Nanobodies, domain antibodies, single domain antibodies or "dAb's", and is most preferably a Nanobody as defined herein.
• the CDR' s may be any suitable CDR' s that provide the desired property to the polypeptide or protein.
• the invention provides one or more of the following main strategies to achieve orally administered polypeptide delivery: a) inhibition of proteolytic activity that degrades polypeptides in stomach and gut, b) developing of protease-resistant polypeptide analogs that retain biological activity, c) stabilizing the polypeptide by conjugation to shielding molecules, d) protecting the polypeptide from proteolytic degradation by e.g. enteric coating, e) improving active (e.g. receptor mediated or M-cell mediated) trans-epithelial transport of the polypeptides, f) increasing half-life of the polypeptide in human body. e.g. at target site, for e.g.
• those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, and/or without being limited to g) improving passive polypeptide transport (diffusion) through the epithelial membrane of the intestine.
• suitable excipient e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, and/or without being limited to g) improving passive polypeptide transport (diffusion) through the epithelial membrane of the intestine.
• the Composition of the Invention may comprise agents that inhibit the proteases (i.e. protease inhibitors) present mainly in the stomach but also to a lesser extend in the gut.
• agents that inhibit the proteases i.e. protease inhibitors
• Such agents are generally known to the skilled person in the ait and may be found in e.g.
• protease inhibitor is an organic acid such as citric or acetic acid.
• Protease inhibitors are readily available for the skilled person in the art.
• Nanobodies or VHH& differed by at most ten amino acid residues from each other and were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid.
• the invention provides pharmaceutical compositions comprising proleolylically stable single variable domains, e.g. Nanobodies or VHHs, wherein said proteolytically stable Nanobodies can be formatted into bi- and/or multivalent (and multimeric) constructs, e.g. into the constructs, polypeptides of the invention.
• polypeptides of the Invention which are conjugated to proteolytically "shielding" molecules such as e.g. pegylated polypeptides comprising single variable domains such as e.g. Nanobodies and/or dAbs.
• the single variable domains e.g. Nanobodies and/or dAbs and constructs described herein may be pegylated. or contain one or more (additional) amino acid residues that allow for pegylation and/or facilitate pegylation.
• Two preferred, but non-limiting examples of such polypeptides are TNF55 and TNF56 as described in WO/2006/122786, which, both contain an additional cysteine residue for easy attachment of a PEG ⁇ group.
• enteric coating that protects the peptide from stomach proteases and which releases active components of the invention in the intestine is suitable.
• the enteric coating functions by providing a coating that does not dissolve in low pH environments, such as the stomach.
• Many enteric coatings are known in the art, and are useful in accordance with the invention. Examples include cellulose acetate phthalate, hydroxypropylmethylethylcellulose succinate, hydroxypropylmethylcellulose phthalate. polyvinyl acetate phthalate, and methacrylic acid- methyl methacrylate copolymer. It is very desirable that all of the active components be released from the dosage form, and solubilized in the intestinal environment as simultaneously as possible. It is preferred that the dosage form release the active components in the small intestine.
• Fc receptors are involved in transcytosis recycling of proteins and other (biological) molecules.
• plgR, FcRn 5 and Vit B12 receptor is known to be involved in transcytosis through biological membranes such as epithelial layers, e.g. in adult human gut.
• 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. single variable domains such as Nanobodies and/or dAbs binding to plgR, FcRn and/or the Vit B12 receptor.
• the building block may also be the natural ligand or fragment of ligand, i.e. human Fc part.
• the building block may also be the natural ligand or fragment of ligand, i.e. human Fc part.
• pharmaceutical compositions comprising the Polypeptides or Constructs of the Invention, wherein said polypeptides 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.
• hGH human growth hormone
• EPO erythropoietin
• Another embodiment of the present invention is a method for selecting Nanobodies, domain antibodies, single domain antibodies or dAbs directed against an epithelial trans-membrane protein, wherein said Nanobody, domain antibody, single domain antibody or dAb crosses the gut membrane upon binding to said epithelial trans-membrane protein.
• Said method comprises panning epithelial trans-membrane protein-displaying membranes with a phage library (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.
• the invention includes a selection method which uses cell lines that over-expresses an epithelial transmembrane protein or cell lines transfected with an epithelial trans-membrane protein gene to allow the easy selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs binding to the epithelial trans-membrane protein. This avoids the need for protein expression and purification, speeding up significantly the generation of membrane crossing Nanobodies, domain antibodies, single domain antibodies or dAbs.
• the invention includes a selection method using cells to allow the selection of phage single variable domains, Nanobodies, domain antibodies, single domain antibodies or dAbs that show receptor mediated internalization. Said method comprises adding the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to the cells and recovering the phage Nanobodies, domain antibodies, single domain antibodies or dAbs from the cells that have undergone internalization.
• the invention includes a selection method using cells seeded on a filter or in a Transwell system or Boyden chamber to allow the selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs that transcytose through the cell monolayer.
• Said method comprises adding the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to compartment 1, allow the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to migrate across the cell monolayer and harvest the phage Nanobodies that migrate in compartment 2.
• the Polypeptides of the Invention comprising e.g. at least a Nanobody or a dAb against a Target Molecule, may also be suitably formulated per se for oral delivery e.g. in the form of a powder (such as a freeze-dried or micronized powder) or mist.
• the Polypeptides of the Invention comprising e.g. at least one Nanobody and/or dAbs, preferably a Nanobody
• the Construct of the Invention is a multispecific polypeptide comprising at least one Nanobody, domain antibody, single domain antibody or dAb directed against a target and at least one Nanobody, domain antibody, single domain antibody or dAb that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific gut membrane cells, or parts or compartments of said cells, and/or that allows the Polypeptide of the Invention to penetrate or cross a biological barrier such as the gut wall or a cell layer of said wall, e.g. membrane.
• Nanobodies, domain antibodies, single domain antibodies or dAbs include Nanobodies, domain antibodies, single domain antibodies or dAbs that are directed towards specific cell-surface proteins, receptors, markers or epitopes of the gut membrane cells.
• the Polypeptides of the Invention comprising e.g. at least one Nanobody and/or dAb, preferably a Nanobody, may comprise one or more Nanobodies, domain antibodies, single domain antibodies or dAbs directed against the desired target and one or more ligand (also called membrane crossing ligand) directed against an epithelial trans- membrane protein on the mucosal membrane, wherein said polypeptide crosses the mucosal membrane upon binding of the ligand to said epithelial trans-membrane protein.
• ligand also called membrane crossing ligand
• An epithelial trans-membrane protein according to the invention is a protein or receptor displayed on the gut membrane which upon binding to a ligand mediates the transport of said ligand through the membrane.
• the ligand is a Polypeptide of the Invention, e.g. a single variable domain, a Nanobody, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine.
• the polypeptide or protein crosses the wall upon binding of said Nanobody, domain antibody, single domain antibody or dAb to said epithelial trans-membrane protein.
• the membrane crossing Nanobody, domain antibody, single domain antibody or dAb may be prepared from a peptide library which is screened for binding to the epithelial trans- membrane protein or for crossing properties.
• Examples of such single variable domains, e.g. Nanobodies, directed against said epithelial trans-membrane protein are the Nanobodies against FcRn, plgR and/or VitB12 receptor as disclosed in the experimental part.
• the Polypeptides of the Invention comprise e.g. at least one single variable domain, a Nanobody and/or a dAb, preferably a Nanobody. and in addition a therapeutic polypeptide or agent, e.g. a Polypeptide of the Invention, e.g. against a Target Molecule, which is covalently or non-covalently linked to said single variable domain, Nanobody, domain antibody, single domain antibody or dAb that is directed against an epithelial trans-membrane protein on the gut membrane. It is an aspect of the invention that these single variable domains, Nanobodies, domain antibodies, single domain antibodies or dAbs can be added as a tag to Polypeptides of the Invention comprising e.g.
• Nanobody and/or a dAb for crossing or passage through the epithelial membrane.
• a therapeutic polypeptide or agent are Nanobodies against FcRn, plgR and/or VitB12 receptor.
• the Polypeptides of the Invention comprise e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, directed against the desired Target Molecule and another ligand (e.g. a natural ligand) of the epithelial trans-membrane protein.
• Another ligand e.g. a natural ligand
• the resulting Polypeptide upon binding of the ligand to the epithelial trans-membrane protein, is transported through the membrane.
• ligand e.g. a natural ligand
• the epithelial trans-membrane protein is the Fc unit or fragment thereof of a human antibody, e.g. the Fc unit of human IgGl.
• the ligand is a Polypeptide of the Invention, e.g. a polypeptide comprising a single variable domain, a Nanobody, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine, and wherein said Polypeptide of the Invention, e.g. a single variable domain, a Nanobody, domain antibody, single domain antibody or dAb directed against an epithelial trans-membrane protein on the gut wall, binds to said trans-membrane protein in a pH dependent manner, preferably binds better at acidic pH, e.g. pH 7 or less, e.g.
• pH dependent single variable domains e.g. Nanobodies
• pH dependent human FcRn and pH dependent human serum albumin binders are exemplified in this application (pH dependent human FcRn and pH dependent human serum albumin binders) and are disclosed in the experimental part.
• the Polypeptides of the Invention comprise e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, directed against the desired Target Molecule and at least another single variable domain, e.g. Nanobody, domain antibody, single domain antibody or dAb that is directed against an epithelial trans-membrane protein on the gut wall, preferably the small intestine, and wherein said other single variable domain, e.g. Nanobody, domain antibody, single domain antibody or dAb binds to said trans-membrane protein in a pH dependent manner, preferably binds better at acidic pH, e.g. pH 7 or less, e.g.
• ligand e.g. a natural ligand
• the epithelial trans-rnembrane protein is transported through the membrane.
• ligand e.g. a natural ligand
• the Fc unit or fragment thereof of a human antibody e.g. the Fc unit of human IgGl .
• a Polypeptide of the Invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention.
• Some preferred, but non-limiting examples of such Polypeptides of the Invention will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or amino acid sequences that is linked to at least one moiety that increases the half-life of the Polypeptide of the
• Polypeptides of the Invention that comprise such half-life extending moieties or amino acid sequences are clear to the skilled person; and for example include, without limitation, polypeptides in which the one or more amino acid sequences are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, single variable domains such as domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, M dAb'"s, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transfen ⁇ ne; reference is made to the further description and references mentioned herein, see e.g.
• the polypeptides 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 polypeptides 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.
• such Polypeptides of the Invention has a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
• such Polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more.
• Polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
• 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 Polypeptides of the Invention or of additional biologically active ingredient(s).
• 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
• Enhancement of passive transport through intestinal gut wall can thus occur by any of a variety of relevant mechanisms, for example by increasing the diffusion, increasing membrane fluidity, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing mucosal membrane components (e.g. lipids), changing non-protein and protein sulfhydryl levels in epithelial tissues, increasing water flux across the surface, modulating epithelial junctional physiology, reducing the viscosity of mucus overlying the epithelium, reducing mucociliary clearance rates, increasing blood flow and other mechanisms.
• Suitable permeability enhancing agents will be clear to a person skilled in the art of pharmacology and are further described hereafter. Such agents may be used in suitable amounts known per se, which will be clear to the skilled person based on the disclosure and prior art cited herein.
• Permeability enhancing agents include (a) aggregation inhibitory agents, (b) charge modifying agents, (c) mucolytic or mucus clearing agents, (d) ciliostatic agents; (f) membrane penetration-enhancing agents such as acyl carnitine, (g) modulatory agents of epithelial junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (h) vasodilator agents, and (i) stabilizing delivery vehicles, carriers, supports or complex-forming species with which the Polypeptide of the Invention is effectively combined, associated, contained, encapsulated or bound to stabilize the Polypeptide of the Invention for enhanced intestinal transport.
• aggregation inhibitory agents include (a) charge modifying agents, (c) mucolytic or mucus clearing agents, (d) ciliostatic agents; (f) membrane penetration-enhancing agents such as acyl carnitine, (g) modulatory agents of epithelial junction physiology, such as
• agents are further exemplified - without being limiting as additional agents comprised in the compositions of the present invention - in WO98034632C2, WO98034632, WO9834632, WO9834632, WO9736480 and/or WO9630036.
• a membrane penetration-enhancing agent is added to the composition of the present invention.
• membrane penetration-enhancing agents have been described such as (i) a surfactant, (ii) a bile salt or bile salt derivative, (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic molecule (viii) a small hydrophobic penetration enhancer, (ix) sodium or a salicylic acid derivative, (x) a glycerol ester of acetoacetic acid, (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium- chain fatty acid, (xiii) a chelating agent (e.g., citric acid, salicylates), (xiv) an amino acid or salt thereof, (xv) an N-
• the membrane penetration-enhancing agent can be selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.
• DMSO dimethyl sulfoxide
• dimethylformamide dimethylformamide
• ethanol propylene glycol
• 2-pyrrolidones 2-pyrrolidones
• long-chain amphipathic molecules for example, deacylmethyl sulfoxide, azone, sodium laiiryl sulfate, oleic acid, and the bile salts (e.g., unsaturated cyclic ureas and Transcutol) may be employed to enhance mucosal penetration of the Nanobodies, polypeptides or proteins of the invention.
• surfactants e.g., Tween 80, Poloxamer 188, polysorbates; further non-limiting examples of surfactants are also provided in EP 490806, US 5,759,565, and WO 04/093917
• These penetration-enhancing agents typically interact at either the polar head groups or the hydrophilic tail regions of molecules that comprise the lipid bilayer of epithelial cells lining the oralmucosa (Barry, Pharmacology of the Skin, Vol. 1, pp. 121-137, Shroot et al.. Eds., Karger, Basel, 1987; and Barry, J.
• the Polypeptide of the Invention is combined with one, two, three, four or more of the permeability enhancing agents recited in (a)-(k) above. These agents may be admixed, alone or together, with the oralcarrier and with the Polypeptide of the Invention, or otherwise combined therewith in a pharmaceutically acceptable formulation or delivery vehicle.
• Nanobodies or dAbs preferably Nanobodies, more preferably agonistic polypeptides, systemic and/or local (i.e. topical gut) delivery
• oral administration by protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art.
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans -epithelial transport of said polypeptides, e.g. by plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.
• active e.g. receptor mediated
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g.
• plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of a suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• a suitable excipient e.g. biodegradable polymer
• the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
• active e.g. receptor mediated
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g.
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g.
• active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• suitable excipient e.g. biodegradable polymer
• covalently binding an unit allowing for longer half life e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) develop protease-resistant polypeptide analogs that retain biological activity, e.g. pharmaceutical oral compositions comprising Target Molecule binding single variable domains, e.g. Nanobodies or dAbs, selected for protease resistance by at least 2. 3, 4, 5 10, 20, 50 100 folds (see e.g. experimental part); c) improving active (e.g.
• receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred IO pM, at pH5 or pH ⁇ or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g.
• a high affinity binding e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred IO pM, at pH5 or pH ⁇ or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g.
• pH dependent plgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and d) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder.
• suitable excipient e.g. biodegradable polymer
• the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
• active e.g. receptor mediated
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g.
• polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g.
• receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g.
• a high affinity binding e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g.
• 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.
• protease inhibitors such as e.g. organic acids
• permeation enhancer such as acylcarnitine and/or Eligen® carrier technology
• Polypeptides of the Invention e.g. Nanobodies or dAbs, preferably Nanobodies
• systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g.
• plgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport preferably plgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and [c) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or d) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcarnitine and/or Eligen® carrier technology].
• protease inhibitors such as e.g. organic acids
• permeation enhancer such as acylcarnitine and/or Eligen® carrier technology
• Nanobodies or dAbs preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) providing continuous local (topical in gut) delivery by bacterial system, e.g. lactic acid bacteria.
• anti-aggregation agents are added to the composition of the invention.
• Aggregation inhibitory agents include, for example, polymers of various functionalities, such as polyethylene glycol, dextran, diethyl aminoethyl dextran, and carboxymethyl cellulose, which significantly increase the stability and reduce the solid-phase aggregation of polypeptides admixed therewith or linked thereto.
• the activity or physical stability of polypeptides can also be enhanced by various additives to pharmaceutical compositions comprising the Polypeptide of the Invention.
• additives such as polyols (including sugars), amino acids, and various salts may be used.
• additives in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized polypeptides.
• These additives can also be used within the invention to protect the polypeptides against aggregation not only during lyophilization but also during storage in the dry state.
• sucrose and Ficoll 70 a polymer with sucrose units
• These additives may also enhance the stability of solid polypeptides embedded within polymer matrices.
• additional additives for example sucrose, stabilize polypeptides against solid-state aggregation in humid atmospheres at elevated temperatures, as may occur in certain sustained-release formulations of the invention.
• 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;118: 11678- 11681), Breslow et al.(PNAS USA 1997; 94: 11156-11158), Breslow et al. (Tetrahedron Lett.
• enzyme inhibitors are added to the composition of the invention.
• the stomach and gut contain hydrolytic enzymes, such as lipases and proteases, which must be overcome.
• This enzymatic "barrier" can be dampened by administering enzyme inhibitors that prevent or at least lessen the extent of degradation.
• Enzyme inhibitors for use within the invention are selected from a wide range of non-protein inhibitors that vary in their degree of potency and toxicity (see, e.g., L. Stryer, Biochemistry, WH: Freeman and Company, NY, NY, 1988). Non-limiting examples include amastatin and bestatin (O'Hagan et al., Pharm. Res. 1990, 7: 112-116).
• enzyme inhibitors are extensively described and exemplified in WO 05/120551 without being limiting for use in the composition of the present invention.
• Another means to inhibit degradation is pegylation with PEG molecules, preferably low molecular weight PEG molecules (e.g. 2 kDa; Lee et al., Calcif Tissue Int. 2003, 73: 545-549).
• PEG molecules preferably low molecular weight PEG molecules (e.g. 2 kDa; Lee et al., Calcif Tissue Int. 2003, 73: 545-549).
• enzyme inhibitor of a Nanobody, domain antibody, single domain antibody or "dAb" directed against said enzyme.
• the invention also relates to a bispecific or multispecific Polypeptide comprising or essentially consisting of one or more Nanobodies, domain antibodies, single domain antibodies or "dAbs” directed against the desired target and one or more Nanobodies, domain antibodies, single domain antibodies or “dAbs” directed against an enzyme of the stomach and/or gut.
• the composition of the invention may further comprise one or more additional therapeutic ingredients (or active substances). These therapeutic ingredients can be any compound that elicits a desired activity or therapeutic or biological response in the subject.
• additional therapeutic ingredients can be any compound that elicits a desired activity or therapeutic or biological response in the subject.
• two or more Nanobodies the invention may be used in combination, i.e. as a combined treatment regimen.
• the pharmaceutical composition of the invention should comprise at least a therapeutically effective amount of the Polypeptide of the Invention, e.g. the polypeptides comprising single variable domains, e.g. Nanobodies.
• a “therapeutically effective amount” as used in the present invention in its broadest sense means an amount of the Polypeptide of the Invention that is capable of eliciting the desired activity or the desired biological, prophylactic and/or therapeutic response.
• the amount of Polypeptide of the Invention to be administered and hence the amount of active ingredient in the pharmaceutical composition of the invention will, of course, vary according to factors such as the bioavailability of the polypeptide, the disease indication and particular status of the subject (e.g., the subject's age, size, fitness, extent of symptoms, susceptibility factors, etc), the target cell, tumor, tissue, graft or organ, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the Polypeptides of the Invention for eliciting the desired activity or biological, prophylactic or therapeutic response in the subject.
• Dosage regimens may be adjusted to provide an optimum activity or biological, prophylactic or therapeutic response. Dosages should also be adjusted based on the release rate of the administered formulation (e.g. a slow release polymer containing composition versus a capsule comprising pressed Polypeptide of the Invention). A therapeutically effective amount is also one in which any toxic or detrimental side effects of the Polypeptide of the Invention are outweighed in clinical terms by therapeutically beneficial effects. Doses may be chosen to be equipotent to the injection route.
• the absolute bioavailability of the Polypeptide of the Invention following oral administration of the Pharmaceutical Composition of the Invention is of the order of ca. 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50. 60, 70, 80, 90, 100% or more of the levels achieved with the corresponding injection.
• Absolute bioavailability measures the availability of the active drug in systemic circulation after oral administration when compared with intravenous administration.
• the absolute bioavailability of the Polypeptides of the Invention is determined by comparing the concentration vs. time plot of the Polypeptides of the Invention after intravenous (IV) administration with the concentration vs. time plot of the Polypeptides of the Invention after oral (IN) administration.
• the absolute bioavailability of Polypeptides of the Invention is defined as (AUC[N x doseiN)/(AUCrv x doseiv) x 100.
• the relative bioavailability of the Polypeptides of the Invention following oral administration of the Pharmaceutical Composition of the Invention is of the order of ca. 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100% or more of the levels achieved with the corresponding injection.
• Relative bioavailability measures the availability of the active drug in systemic circulation after oral administration when compared with another form of administration of the same drug, such as intramuscular (IM) or subcutaneous (SC).
• IM intramuscular
• SC subcutaneous
• the amount of active compound will generally be chosen to provide effective treatment on administration once a day or once a week or once a month. Alternatively, dosages may be split over a series of e.g. 1 to 4 applications taken at intervals during the day, week or month. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple takings of a plurality of pills or capsules.
• the Composition of the Invention is repeatedly administered to the subject, for example one, two or more times within a 24 hour period, four or more times within a 24 hour period, six or more times within a 24 hour period, or eight or more times within a 24 hour period.
• An administration regimen could include long-term, daily, weekly or monthly treatment.
• long-term is meant at least two weeks and preferably, several weeks, months, or years of duration. The clinician will generally be able to determine a suitable daily, weekly or monthly dose, depending on the factors mentioned herein.
• the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.
• the final determination of the effective dosage will be based on animal model studies, followed up by human clinical trials, and is guided by determining effective dosages and oral administration protocols that significantly reduce the occurrence or severity of the targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art.
• the dosage of Polypeptides of the Invention will be at the discretion of the attendant, physician or clinician. The dosage can also be adjusted by the individual physician in the event of any complication.
• the Polypeptides of the Invention is suitably presented in the Pharmaceutical Composition of the Invention in an amount such as to provide a free Polypeptides of the Invention concentration from about 0.1 microgram to 0.1 gram per kg body weight per day, such as from 1 microgram to 0.1 gram per kg body weight per day, such as from 0.01 to 100 milligram per kg body weight per day, such as from 0.05-100 milligram, such as from 0.05 to 50 milligram, 0.05 to 30 milligram, 0.1 to 20 milligram, or from about 1 to 10 or about 5 to 10 milligram per kg body weight per day either as a single daily dose or as multiple divided doses during the day.
• each further component in the oral composition of the invention may vary depending on the components used.
• the amount of enteric coating may be in the range of from 0.1 to 99.9 %, preferably 1 to 20% by weight of the total weight of the composition.
• the amount of permeability enhancer may be in the range from about 0.01 to about 10% or higher and preferably about 0.05 to about 1.0% by weight of the total weight of the composition, the amount depending on the specific enhancer used. The amount is generally kept as low as possible since above a certain level no further enhancement of absorption can be achieved and also too high of a enhancer level may cause irritation of the gut.
• the amount of protease inhibitor may be at least 0.1%, suitably in the range from about 0.5 to 10 % of the total weight of the composition. Preserving agents may be present in an amount of from about 0.002 to 0.02 % by weight of the total weight or volume of the composition.
• the amount of the other excipients will be determined by processes known to the skilled person in the art.
• the total delivery weight is important to consider as well.
• the delivery weight is relatively high for oral compositions and may be up to 1 g or more. Suitable delivery weights will be clear to a person skilled in the art of pharmacology.
• 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 Polypeptides of the Invention and the excipients are milled into smaller granules (dry or wet granulation) and pressed into the core pill - said core pill is then further coated e.g. by enteric coating. All above described steps may be prepared in a conventional manner known to the skilled person in pharmacology.
• the solid oral composition of the invention may be prepared in conventional manner.
• the Polypeptides of the Invention e.g. Nanobodies
• the Polypeptides of the Invention e.g.
• Nanobodies may be in solution e.g. an aqueous or alcoholic solution when being mixed with the protease inhibitors, slow release matrices, and/or permeability enhancer and the solvent evaporated, e.g. under freeze-drying or spray drying. Such drying may be effected under the conventional conditions. Alternatively the dry mixtures may be compacted and/or granulated and then be pulverized and/or sieved. If desired the compacted composition may be further coated. According to a preferred embodiment of the invention, the oral composition is prepared by lyophilisation, then granulated and filled up into enterically coated capsules. A homogeneous solution, preferably aqueous, containing the Polypeptides of the Invention, e.g.
• Nanobodies and optionally containing further ingredients, additives and/or agents as discussed above, e.g. protease inhibitors, slow release matrices, and/or permeability enhancer, is prepared and then submitted to lyophilisation in analogy with known lyophilisation procedures, and to subsequent drying.
• the resulting powder may then be filled up into enterically coated capsules before administration.
• the Polypeptides of the Invention may be administered in liquid form such as in the form of a suspension or partly or fully dissolved solution, e.g. the lyophilized powder may be reconstituted in e.g. water before administration or may be stored in liquid form and thus may be directly be used as such.
• compositions For administration of a liquid, for example, such compositions will suitably be put up in a container provided with a conventional dropper/closure device, e.g. comprising a pipette or the like, preferably delivering a substantially fixed volume of composition/drop.
• a conventional dropper/closure device e.g. comprising a pipette or the like, preferably delivering a substantially fixed volume of composition/drop.
• a powder or liquid may be filled into a soft or hard capsule adapted for oral administration.
• the powder may be sieved before filled into the capsules such as gelatine capsules, preferably an enterically coated capsule.
• the Pharmaceutical Composition of the Invention is formulated for oral administration and for delivery of the Polypeptides of the Invention (at least the therapeutically active moiety) either locally to the gut and/or systemically to the body providing a systemic therapeutic or biological response of the Polypeptides of the Invention, e.g. the Nanobodies, in the subject.
• the bioavailability of the Polypeptides of the Invention, e.g. the Nanobodies, in the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) and/or in the brain following administration of the composition of the invention is determined by measuring the pharmacokinetic parameters Cmax (peak concentration), AUC (area under concentration vs. time curve) and/or Tmax (time to maximal blood concentration), which are well known to those skilled in the art (Laursen et al., Eur. J.
• the bioavailability of the Polypeptide of the Invention may be determined in any conventional manner, e.g. by radioimmunoassay.
• Cmax is the mean maximum concentration of the Polypeptide of the Invention achieved in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung), following oral administration of a single dosage of the pharmaceutical composition to the subject.
• Blood or bloodstream as used in the present invention, can be any form and/or fraction of blood. Without being limiting, blood or bloodstream includes plasma and/or serum.
• the Cmax for the Polypeptide of the Invention comprised in the pharmaceutical composition of the invention can have any value as long as said Polypeptide of the Invention provides the desired activity or therapeutic or biological response in the subject in need of said Polypeptide of the Invention, e.g. the Nanobodies.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention per ml of blood.
• the Polypeptide 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 Polypeptide of the Invention, e.g. the Nanobodies, per ml of blood.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention, per ml of blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention, e.g. the Nanobodies.
• the Polypeptide 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 Polypeptide of the Invention, per ml of blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
• said polypeptide following oral administration of Polypeptide of the Invention, said polypeptide reaches a Cmax in blood of at least 1% of the Craax that is reached following parenteral administration of the same amount of the Polypeptide of the Invention. In a further embodiment, following oral administration of the Polypeptide of the Invention, said Polypeptide 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 Polypeptide of the Invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) following oral administration of a single dosage of the composition of the invention.
• the Tmax for the Polypeptide of the Invention comprised in the composition of the invention can have any value as long as said Polypeptide of the Invention provides the desired activity or therapeutic or biological response in the subject in need of said Polypeptide of the Invention.
• the Polypeptide of the Invention reaches the bloodstream with a Tmax of less than 120 minutes.
• the Polypeptide of the Invention reaches the bloodstream with a Tmax of less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes. In a further embodiment, the Polypeptide of the Invention reaches the brain with a Tmax of less than 90. 60, 50, 40, 30, 20, 10, or 5 minutes.
• the "concentration vs. time curve” measures the concentration of the Polypeptide of the Invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) of a subject vs. time after administration of a dosage of the composition of the invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention per ml of blood within less than 120 minutes following oral administration of the composition of the invention. In a further embodiment, the Polypeptide of the Invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of Polypeptide of the Invention per ml of blood within less than 120 minutes following oral administration of the composition of the invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention per ml of blood within less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of the composition of the invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100. 150. 200, 300, 400, 500, 750, 1000 ng or more of Polypeptide of the Invention per ml of blood within less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of the composition of the invention,
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of Polypeptide of the Invention per ml of blood within less than 120 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 3O 5 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of the Polypeptide of the Invention per ml of blood within less than 120 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 1 ng of the Polypeptide of the Invention per ml of blood within less than 90, 60. 50. 40, 30, 20, 10, or 5 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
• the Polypeptide of the Invention reaches a Cmax in blood of at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 750, 1000 ng or more of Polypeptide of the Invention per ml of blood within less than 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
• AUC area under the curve
• AUC is the area under the curve in a plot of concentration of the Polypeptide of the Invention in blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) against time.
• this value is a measure of the integral of the instantaneous concentrations during a time interval.
• AUC is usually given for the time interval zero to infinity, and other time intervals are indicated (for example AUC (ti,t 2 ) where t] and t 2 are the starting and finishing times for the interval).
• blood (and/or another selected physiological compartment, tissue and/or organ such as e.g.
• Polypeptide of the Invention concentrations cannot be measured 'to infinity " for a subject so mathematical approaches are used to estimate the AUC from a limited number of concentration measurements.
• the AUC (from zero to infinity) is used to measure the total amount of Polypeptide of the Invention absorbed by the body, irrespective of the rate of absorption, This is useful when trying to determine whether two application formulations with the same dose (for example parenteral and oral) release the same dose of Polypeptide of the Invention to the body.
• the AUC for the Polypeptide of the Invention comprised in the composition of the invention can have any value as long as said Polypeptide 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 Polypeptide of the Invention in blood following oral administration of a composition comprising said Polypeptide of the Invention is at least 500 ng/ml/minute of the Polypeptide of the Invention.
• the AUC for the Polypeptide of the Invention in blood following oral administration of a composition comprising said Polypeptide 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 Polypeptide of the Invention.
• the AUC for the Polypeptide of the Invention in blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention is at least 500 ng/ml/minute Polypeptide of the Invention.
• the AUC for the Polypeptide of the Invention in blood following oral administration of a dose of 5 mg/kg body weight of said Polypeptide 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 Polypeptide of the Invention per ml of blood.
• the bioavailability (absolute or relative) for the Polypeptide of the Invention in blood following oral administration of a composition comprising said Polypeptide of the Invention is at least 1% compared to parenteral administration of said Polypeptide of the Invention.
• the bioavailability for the Polypeptide of the Invention in blood following oral administration of a composition comprising said Polypeptide of the Invention is at least 2, 3, 5, 7, 10, 15, 20, 25, 30. 40, 50. 60, 70, 80, 90, 100% or more compared to parenteral administration of said Polypeptide of the Invention.
• the bioavailability (absolute or relative) for the Polypeptide of the Invention in blood following oral administration of a composition comprising said Polypeptide of the Invention is at least 5% compared to parenteral administration of said Polypeptide of the Invention.
• Oral administration of one or more Polypeptides of the Invention to a subject yields effective delivery of the Polypeptides of the Invention to the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) to elicit the desired activity or therapeutic or biological response in the subject.
• the Polypeptide of the Invention provides the prevention and/or treatment of a selected disease or condition in said subject.
• another aspect of the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention, said method comprising orally administering, to said subject a therapeutically effective amount of said Polypeptide of the Invention, and/or of a composition comprising the same.
• prevention and/or treatment not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.
• the subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and/or disorder.
• the invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a Polypeptide of the Invention to a subject suffering from said disease or disorder, said method comprising orally administering to said subject a therapeutically effective amount of the Polypeptide of the Invention, and/or of a composition comprising the same. Accordingly, the invention relates to the Polypeptides or compositions of the invention for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention.
• the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises oral administering, to a subject suffering from or at risk of a diseases and/or disorders that can be cured or alleviated by immunotherapy with a Polypeptide of the Invention, a therapeutically effective amount of said Polypeptide of the Invention and/or of a composition comprising the same.
• the polypeptides present in the compositions of the invention may be directed against any suitable target that is of therapeutic or diagnostic interest.
• the polypeptides can be functional as agonists as well as antagonists, preferably agonists. Examples include but are not limited to targets of therapeutic interests such as EPO, Growth Hormone, TNF- ⁇ , IgE, IFN - ⁇ , MMP- 12, EGFR, CEA, H. pylori, M. tuberculosis, influenza, ⁇ -amyloid, vWF, IL-6, IL-6R, PDKl, CD40, OVA, VSG, S. typhimurium, Rotavirus, Brucella, parathyroid hormone-derived peptides.
• targets of therapeutic interests such as EPO, Growth Hormone, TNF- ⁇ , IgE, IFN - ⁇ , MMP- 12, EGFR, CEA, H. pylori, M. tuberculosis, influenza, ⁇ -amyloid, vWF
• the invention provides systemic delivery of the Polypeptide of the Invention.
• the desired target can be a target in any physiological compartment, tissue or organ.
• the Polypeptide of the Invention is directed against a target in the kidney or the bladder and the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention that is directed against a target in the kidney or bladder, said method comprising orally administering, to said subject a therapeutically effective amount of said Polypeptide 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 Polypeptide 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 Polypeptide 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 Polypeptide 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 Polypeptide 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 Polypeptide of the Invention is directed against a target in the lung and the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention that is directed against a target in the lung, said method comprising orally administering, to said subject a therapeutically effective amount of said Polypeptide 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 Polypeptide of the Invention that is directed against a target in the lung, said method comprising orally administering to said subject a therapeutically effective amount of said Polypeptide of the Invention, and/or of a composition comprising the same.
• the invention also relates to a method for the prevention and/or treatment of a disease or disorder of the lung, said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of the Invention that is directed against a target in the lung and/or of a composition comprising the same.
• the invention also relates to the composition of the invention, wherein the Polypeptide of the Invention is directed against a target in the lung for the prevention and/or treatment of at least one disease or disorder of the lung.
• the Polypeptide 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 Polypeptide 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 Polypeptide 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 Polypeptide 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 Polypeptide of the Invention, and/or of a composition comprising the same.
• the invention also relates to a method for the prevention and/or treatment of a tumor related disease or disorder, said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of the Invention that is directed against a target on a tumor and/or of a composition comprising the same.
• the invention also relates to the composition of the invention, wherein the Polypeptide of the Invention is directed against a target on a tumor for the prevention and/or treatment of at least one a tumor related disease or disorder.
• the Polypeptide of the Invention is directed against TNF and the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention that is directed against TNF 3 said method comprising orally administering, to said subject a therapeutically effective amount of said Polypeptide 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 Polypeptide of the Invention that is directed against TNF, said method comprising orally administering to said subject a therapeutically effective amount of said Polypeptide of the Invention, and/or of a composition comprising the same.
• a disease or disorder such as an autoimmune disease (such as e.g.
• the present invention also relates to the composition of the invention, wherein the Polypeptide of the Invention is directed against TNF for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease).
• an autoimmune disease such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease
• the Polypeptide of the Invention is directed against vWF and the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention that is directed against vWF, said method comprising orally administering, to said subject, a therapeutically effective amount of said Polypeptide 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 Polypeptide of the Invention that is directed against vWF, said method comprising orally administering to said subject a therapeutically effective amount of said Polypeptide 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.
• 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), said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of the Invention that
• the present invention also relates to the composition of the invention, wherein the Polypeptide of the Invention is directed against vWF for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g.
• 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
• the Polypeptide of the Invention is directed against IL-6, IL-6R and/or IL-6/IL-6R complex and the invention relates to a method for the prevention and/or treatment of a subject in need of a Polypeptide of the Invention that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering, to said subject, a therapeutically effective amount of said Polypeptide 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 Polypeptide of the Invention that is directed against IL-6, 1L-6R and/or IL-6/IL-6R complex, said method comprising orally administering to said subject a therapeutically effective amount of said Polypeptide 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 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), said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of
• the present invention also relates to the composition of the invention, wherein the Polypeptide of the Invention is directed against IL- 6, IL-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).
• the Polypeptides of the Invention and/or the compositions comprising the same are orally administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated.
• the clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific Polypeptide of the Invention to be used and the pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the subject, and similar factors well known to the clinician.
• the treatment regimen will comprise the oraladministration of one or more Nanobodies, polypeptides or proteins of the invention, or of one or more compositions comprising the same, in one or more therapeutically effective amounts or doses.
• the specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.
• Nanobodies and polypeptides 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.
• a second active substances or principles When a second active substances or principles is to be used as part of a combined treatment regimen, it can be administered via the same oralroute of administration or via a different route of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime).
• the substances or principles When the substances or principles are administered to be simultaneously via the same oralroute of administration, they may be administered as different formulations or compositions or part of a combined formulation or composition, as will be clear to the skilled person.
• 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 effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician.
• the clinician will also be able, where appropriate and or a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.
• the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.
• the invention also relates to the use of a Polypeptide 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 Polypeptide 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 Polypeptide of the Invention directed against a target in the kidney or the bladder.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target in the kidney or the bladder for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the kidney or bladder.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target in the lung for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against a target in the lung.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target in the lung for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the lung.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target on a tumor for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against a target on a tumor.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target on a tumor for the preparation of a composition for the prevention and/or treatment of at least one cancer.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target in the brain for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against a target in the brain.
• the invention also relates to the use of a Polypeptide of the Invention directed against a target in the brain for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder of the brain (such as neurogenetic diseases, (e.g. Huntington's disease and muscular dystrophy), developmental disorders (e.g. cerebral palsy), degenerative diseases of adult life (e.g.
• Parkinson's disease and Alzheimer's disease metabolic diseases (e.g. Gaucher' s disease), cerebrovascular diseases (e.g. stroke and vascular dementia), trauma (e.g. spinal cord and head injury), convulsive disorders (e.g. Epilepsy) infectious diseases (e.g. AIDS dementia), obesity, diabetes, anorexia, depression, brain tumors, dementia with Lewy bodies, multi-system atrophy, progressive supranuclear palsy, frontotemporal dementia, vascular dementia or Down's syndrome).
• metabolic diseases e.g. Gaucher' s disease
• cerebrovascular diseases e.g. stroke and vascular dementia
• trauma e.g. spinal cord and head injury
• convulsive disorders e.g. Epilepsy
• infectious diseases e.g. AIDS dementia
• obesity diabetes, anorexia, depression, brain tumors, dementia with Lewy bodies, multi-system atrophy, progressive supranuclear palsy, frontotemporal dementia, vascular dementia or Down's syndrome.
• the invention also relates to the use of a Polypeptide of the Invention directed against TNF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against TNF.
• the invention also relates to the use of a Polypeptide of the Invention directed against TNF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease).
• the invention also relates to the use of a Polypeptide of the Invention directed against vWF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against vWF.
• the invention also relates to the use of a Polypeptide of the Invention directed against vWF for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g.
• 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
• the invention also relates to the use of a Polypeptide of the Invention directed against IL- ⁇ , IL-6R and/or IL-6/IL-6R complex for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by orally administering to a subject a Polypeptide of the Invention directed against IL- 6, IL-6R and/or IL-6/IL-6R complex.
• the invention also relates to the use of a Polypeptide of the Invention directed against IL-6, IL-6R and/or IL-6/IL-6R complex for the preparation of a composition for the prevention and/or treatment of at least one disease or disorder associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC).
• MM multiple myeloma disease
• RCC renal cell carcinoma
• BLPD B-iymphoproliferative 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).
• BLPD B-iymphoproliferative disorder
• cachexia psoriasis
• mesangial proliferative glomerulonephritis Kaposi's sarcoma
• oral administration of one or more Polypeptides of the Invention to a subject yields effective delivery of the Polypeptides of the Invention to the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) and/or to the brain to elicit the desired activity or biological response in the subject.
• the Nanobodies, polypeptides and proteins of the invention may also induce other activities and biological responses.
• the present invention also provides for the diagnostic use of the Polypeptides of the Invention, e.g. for in situ or in vivo labeling, such as radiolabeling and imaging.
• the present invention therefore, also relates to a diagnostic method comprising the step of orally administering the Polypeptides of the Inventionand/or a composition comprising the same.
• a diagnostic method comprising the steps of orally administering the Polypeptides of the Invention and/or a composition comprising the same and in situ detecting said Polypeptides of the Invention. Detection may be done by any method known in the art.
• the Polypeptides of the Invention can be determined in situ by non-invasive methods including but not limited to SPECT and PET, or imaging methods described by Cortez- Retamozo V. (Nanobodies: single domain antibody fragments as imaging agents and modular building blocks for therapeutics, PhD Dissertation, Vrije Universiteit Brussel, Belgium, June 2004), Arbit et al. (Eur. J. Nucl. Med. 1995; 22: 419-426.), Tamada et al. (Microbiol- Immunol. 1995; 39: 861-871), Wakabayashi et al. (Noshuyo-Byori 1995; 12: 105-110), Huang et al. (Clin. Med. J.
• the Polypeptide of the Invention orally administered in the diagnostic methods of the invention may be labeled by an appropriate label.
• the particular label or detectable group used in the method is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the Polypeptide of the Invention used in the method.
• the detectable group can be any material having a detectable physical or chemical property.
• detectable labels have been well developed in the field of immunoassays and, in general, almost any label useful in such methods can be applied to the method of the present invention.
• a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means.
• Useful labels in the present invention include but are not limited to magnetic beads (e.g.
• DynabeadsTM DynabeadsTM
• fluorescent dyes e.g. fluorescein isothiocyanate, Texas red, rhodamine, Cy3, Cy5 5 CyS.5, Alexi 647 and derivatives
• radiolabels e.g. 3 H, 125 1, 35 S, 14 C, 32 P or 99m Tc
• enzymes e.g. horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA
• colorimetric labels such as colloidal gold, colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
• the label may be coupled directly or indirectly to the Polypeptide of the Invention according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, the available instrumentation and disposal provisions. Non-radioactive labels are often attached by indirect means. Means for detecting labels are well known in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of a photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
• CCDs charge coupled devices
• EpoR erythropoietin receptor
• GHR growth hormone receptor
• the GHR dimerization and signaling induced by the growth hormone is the key regulator of postnatal growth and has important actions on metabolism, reproductive, gastrointestinal, cardiovascular, hepto-biliary and renal systems (Brooks et al, 2007). Because of the existence of many clinical situations where the circulating red blood cell levels are reduced provoking anemia, some efforts have been made to develop stable and potent erythropoietin mimetic peptides (EMPs) that activate the receptor by dimerization and thus mimic Epo action.
• EMPs erythropoietin mimetic peptides
• EpoR agonist Some bivalent monoclonal antibodies have been described as EpoR agonist since they are capable of forming receptor dimers and stimulate cell proliferation in EpoR-expressing cells, while monovalent Fab fragments fail (Wrighton et al., 1996; Schneider et al., 1997; Skelton et al, 2002; Vadas and Rose, 2007).
• agonist monoclonal antibodies For the related GH receptor, a variety of agonist monoclonal antibodies have been also reported (RowJinson et al., 1998).
• EpoR and GHR as other members of the cytokine receptor type I superfamily. are cell surface proteins composed of an NH 2 -terminal ligand binding domain, a COOH-terminal cytoplasmic region and a single membrane-spanning domain. conserveed features of the extracellular domain include two pairs of cysteine residues and a 'WSXWS" motif with characteristic spacing.
• GHR The classical model for activation of GHR is described as the formation of a ligand-receptor complex made up of one GH molecule and two GHR (GH:2GHR).
• GH:2GHR One of the monomer receptors binds with a strong affinity to site 1 of the GH followed by the weaker site 2 binding to the second receptor (Watowich, 1999; Brooks et al., 2007).
• Epo Reduction of red blood cell levels by a failure in the Epo synthesis provoking anemia is associated to many pathological conditions including chronic renal failure, malignancy or the effects of chemotherapy used to treat cancer, HIV and rheumatoid arthritis (Watowich, 1999. Review. Kontantinopoulos et al., 2007). So Epo is used normally therapeutically administered either by intravenous or subcutaneous injection. However the fact that Epo is large glycoproteins has a negative impact on the cost of the manufacture and on the mode of delivery of this therapeutic agent. Therefore the development of new molecules that can mimic the Epo trough interaction with EpoR is clearly envisaged.
• GH has been of significant scientific interest for decades because of its capacity to dramatically change physiological growth parameters.
• GH has been used for the treatment of adults with GH deficiency and conditions such as Turner ' s syndrome, Prader- Willie syndrome, intrateurine growth restriction and chronic renal failure (Dattani and Preece, 2004). Mutations in the GHR have been described as the cause of the Laron Syndrome that is characterised by severe postnatal growth retardation (Rosenfeld et al., 1994).
• Example 2 Library construction Peripheral blood mononuclear cells will be prepared from blood samples using Ficoll- Hypaque according to the manufacturer's instructions. Next, total RNA will be extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments will be cloned into phagemid vector pAX50. Phage will be prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
• Phage libraries 215 and 216 will be used for selections on recombinant mouse EpoR/Fc Chimera (R&D Systems Cat No 1390-ER). rm EpoR/Fc will be immobilized directly or captured by an anti human Fc antibody on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). To minimize the number of phage binding to the Fc- portion of EpoR/Fc the phage will be pre-incubated with 250 ug/ml human IgG.
• the Phage libraries will be pre-incubated with jejunal or gastric fluid prior to selection (analog to Harmsen, 2006, supra) in order to select for protease -resistant Nanobodies.
• Phage libraries will be selected for EpoR binding in the presence of jejunal or gastric fluid (again pre-incubated and not pre-incubated).
• the clones will be 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) will be tested. Shortly, 0.2 ug/ml receptor will be immobilized on Maxisorp ELISA plates (Nunc) and free binding sites will be 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 will be allowed to bind to the immobilized antigen.
• phage binding will be revealed using a HRP-conjugated monoclonal -anti -M 13 antibody (Gentaur Cat# 27942101). Binding specificity will be determined based on OD values compared to controls having received no phage and to controls where in a similar ELISA binding assay the same monoclonal phage will be tested for binding to 0.2 ug/ml of immobilized human IgG.
• Example 5 Screening for Nanobodies competing or non-competing (non-neutralizing) Epo-EpoR interaction.
• EpoR binding assay Clones tested positive in the EpoR binding assay (including those selected for protease resistancy) will be screened for their ability to block Epo binding to EpoR/Fc.
• positive binding EpoR phage will be used in an ELISA-based ligand competition setup. 10 ul of supernatant from the monoclonal phage inductions of the different positives clones will be mixed with increasing amounts of EPO and added to 96 well Maxisorp microtiter plates (Nunc) coated with EpoR. After incubation and washing steps, phage binding will be revealed using a HRP -conjugated monoclonal-anti-M13 antibody (Gentaur Cat# 27942101).
• Binding specificity will be determined based on OD values compared to controls having received no Epo and/or no phage.
• the same kind of competition assay could be performed using Nanobody-containmg periplasmic extracts (P. E.) instead of phage and detecting with a mouse anti-myc antibody and an anti mouse-HRP antibody.
• EpoR binding assay Clones tested positive in the EpoR binding assay (including clones selected for protease resistancy) will be screened for their ability do not to block EpoR binding to EpoR/Fc.
• positive binding EpoR phage will be used in an ELISA-based ligand competition setup. 10 ul of supernatant from the monoclonal phage inductions of the different positives clones will be mixed with increasing amounts of EpoR and added to 96 well Maxisorp microtiter plates (Nunc) coated with EpoR. After incubation, eluted phage containing non-neutralizing Nanobodies will be further analyzed e.g. in BioCore experiments and verified whether indeed they are non-neutralizing Nanobodies.
• these non-neutralizing Nanobodies will be used preferably for the construction of agonistic construct comprising e.g. 2 EpoR non- neutralizing Nanobodies.
• various constructs will be generated comprising 2 EpoR non-neutralizing Nanobodies that are identified above, e.g. linked by a 9 GIy linker (see e.g. WO 2007/104529).
• Example 5a Testing Screened non-competing Nanobodies (non-neutralizing) for EpoR agonism using MAPPIT (J. Tavernier et al., MAPPIT: a cytokine receptor-based two- hybrid method in mammalian cells, CHn. Exp. Allergy 2002; 32: 1397-1404).
• MAPPIT a cytokine receptor-based two- hybrid method in mammalian cells, CHn. Exp. Allergy 2002; 32: 1397-1404.
• a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric "bait" construct, e.g.
• Peripheral blood mononuclear cells will be prepared from blood samples using Ficoll- Hypaque according to the manufacturer's instructions. Next, total RNA will be extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments will be cloned into phagemid vector pAX50, Phage will be prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
• Phage libraries 215 and 216 will be used for selections on recombinant mouse GHR/Fc Chimera (R&D Systems Cat No 1360-GR). rm GHR/Fc will be immobilized directly or captured by an anti human Fc antibody on Maxisorp 96 well microliter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). To minimize the number of phage binding to the Fc- portion of GHR/Fc the phage will be pre-incubated with 250 ug/ml human IgG.
• the Phage libraries will be pre-incubated with jejunal or gastric fluid prior to selection (analog to Harmsen, 2006, supra) in order to select for prolcase-resistant Nartobodies. Based on preliminary reports we will chose in one arm a GI fluid concentration that resulted in a decrease in antigen binding capacity in phage ELISA to 10% of an untreated control. In another arm, the Phage libraries will be selected for EpoR binding in the presence of jejunal or gastric fluid (again pre-incubated and not pre-incubated).
• Example 9 Screening for Nanobodies competing GH-GHR interaction or not competing GH-GHR Clones tested positive in the GHR binding assay (including clones selected for protease resistancy) will be screened for their ability to block GH binding to GHR/Fc. For this, positive binding GHR phage will be used in an ELISA-based ligand competition setup.
• Clones tested positive in the GHR binding assay will be screened for their ability not to block GH binding to GHR/Fc.
• positive binding GHR phage will be used in an ELISA-based ligand competition setup. 10 ul of supernatant from the monoclonal phage inductions of the different positives clones will be mixed with increasing amounts of GH and added to 96 well Maxisorp microtiter plates (Nunc) coated with GHR. After incubation, eluted phage containing non-neutralizing Nanobodies will be further analyzed e.g. in BioCore experiments and verified whether indeed they are non-neutralizing Nanobodies.
• these non-neutralizing Nanobodies will be used preferably for the construction of agonistic construct comprising e.g. 2 GHR non- neutralizing Nanobodies.
• various constructs will be 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 9a Testing Screened non-competing Nanobodies (non-neutralizing) for identifying GHR agonism using MAPPIT (J. Tavernier et al., MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells, CHn. Exp.
• a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric "bait" construct, e.g.
• Watowich SS Watowich SS.
• the major histocompatibility complex class I-related receptor FcRn was first identified as the receptor that transports maternal IgGs from mother to young via the neonatal intestine.
• the neonatal receptor is also responsible for rescuing IgG and albumin from degradation and therefore prolong their half-lives (Andersen el al, 2006; Anderson et al, 2006; Ghetie and Ward, 2000; Kim et al.. 2006; Lencer and Blumberg, 2005; Ober et al., 2004a; Ober et al., 2004b).
• FcRn is expressed inside endothelial cells that line blood vessel, mainly in early/recycling endosomes, where IgG and albumin can be internalized by fluid phase endocytosis. To a minor extend FcRn is also express in the cell surface. IgG and albumin bind independently to FcRn in a pH-dependent manner, with binding at pH 6.0 but not at pH 7.4. The acidic environment of the endosomes facilitates the interaction. Bound IgG and albumin are recycled back to the surface and released from the cell, while unbound ligands are shuttled downstream to lysosomal degradation (Ghetie and Ward, 2000).
• FcRn as an IgG transporter opens the opportunity to generate new therapeutics for modulation of IgG levels, as it is desired in the case of autoimmune diseases. Because the transport and protection of IgG are dependent on its Fc-domain, it can be proposed that small molecules or peptides with therapeutic activities could be fused to Fc fragments and therefore delivered across the epithelium and have long circulating long lives. Moreover, the fact that FcRn is expressed on many epithelial surfaces in adult humans including the lungs
• FcRn transport pathway could be used as a delivery system of therapeutic agents by non-invasive means (i.e. aerosols administered into the lungs using normal breathing maneuvers).
• FcRn comprises a heterodimer of beta2 -micro globulin and a 45 to 53 KDa protein. All three extracellular and membrane domains of FcRn share homology with the corresponding regions of major histocompatibility complex (MHC) class I molecules, with much less homology between the cytoplasmic domains.
• MHC major histocompatibility complex
• the X-ray crystallographic structure of the extracellular domains of FcRn confirmed that it is structurally similar to MHC class I molecules (Ghetie and Ward, 2000).
• the FcRn-IgG interaction depends on conserved histidine residues in the IgG-Fc part that interact with negatively charged residues in the beta-2 domain of the hFcRn heavy chain. Recent studies showed that conserved H 166 in the hFcRn heavy chain, directly opposite to the IgG binding site, is a key player in the FcRn-albumin interaction (Andersen et al., 2006).
• FcRn regulates IgG homeostasis, modulation of FcRn function and/or expression might be an effective approach for the treatment of autoimmune diseases. It has been suggested that deregulation of FcRn expression may be involved in situations in which hypercatabolism is observed, such as after burns and in myotome dystrophy. It is also possible that some types of IgG deficiencies such as familial idiophatic hypercatabolism may be caused by abnormalities in FcRn expression or function (Ghetie and Ward, 2000).
• LG215-C3 evqlvesggglvqpggslrlscaasgftfsDHAMSwvrqapgkglewvs,.
• AINTAGIIT LG215-E2 evqlvesggglvqpggsMscaasgftfsDYAMSwvrqapgkglewvs..
• AINTAGIIT LG215-B5 evqlvesggglvqpggslrlscaasgftfsD YAMS wvrqapgkglewvs..
• LG215 -B5 NYADSVKGrftmsrdnakntlylqmnslkpedtgkyycar .NRDINNSRPP esqg LG215-B3
• LG216-E1 1 tqvtvss
• LG215-A4 tqvtvss
• Llama 153 was immunized, according to standard protocols, with 6 boosts of a cocktail 112 containing hFcRn HC (only the human FcRn heavy chain).
• hFcRn HC and shFcRn mutmix were kindly provided by Inger Sandlie (University of Oslo, Norway).
• Llama 154 was immunized, according to standard protocols, with 3 boosts of a cocktail 116 containing shFcRn mutraix (intact soluble FcRn, heavy chain and beta2- microglobulin).
• Peripheral blood mononuclear cells were prepared from blood samples using FicoU-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
• Example 12 Selections of phage displaying hFcRii binding Nanobodies Phage library 153 was used for selection at pH 5 on hFcRn heavy chain (hFcRn HC) while phage library 154 was used for selection at pH 5 on shFcRn (heavy chain and beta2- microglobulin).Both hFcRn proteins were immobilized directly on Maxisorp 96 well microliter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control) in PBS at pH 7.4.
• the eluted phage were amplified and applied in a second round of pH 5 selection on 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control) immobilized hFcRn proteins.
• the phage was pre-incubated with 250 ug/ml human serum albumin in 2 % Marvel PCA buffer pH 5.1.
• Individual colonies obtained from the eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for " Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).
• Example 13 Screening for hPeRn 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 hPcRn HC was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. After washing with PCA ph 5.1 buffer, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PCA pH 5.1 were allowed to bind to the immobilized antigen.
• Lencer WI and BJumberg RS Lencer WI and BJumberg RS.
• Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4 0 C for further use.
• Example 16 Selections Phage libraries from llama's 097 and 098 were used for selections for two rounds on ectodomain of plgR (R&D Systems Cat # 2717-PG).
• plgR was immobilized directly on Nunc Maxisorp ELISA plates at 5 micro g/ml or 1 ug/ml and 0 ug/ml (low control) for the first round of selection and 5 microg/ml or 0.5 ug/ml and 0 ug/ml (low control) for the second round of selection.
• Binding phages were retrieved from both first and second selection rounds using trypsin ⁇ lution, IgA specific elution and BSA specific elution (neg. control).
• the clones were tested in an ELISA binding assay setup.
• 5 ug/rnl plgR ectodomain was immobilized on Maxisorp microtiter 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).
• Example 18 screening for competition
• Example 19 determining competition efficiency by titration of purified Nanobody
• 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 After incubation and a wash step, IgA binding was revealed using a rabbit-anti- IgA secondary antibody (Serotec cat# AHP525H). which was after a wash step detected with a HRP-conjugated donkey-anti-rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control) and two Nanobodies that can bind to plgR but do not compete for IgA binding.
• a rabbit-anti- IgA secondary antibody Serotec cat# AHP525H
• HRP-conjugated donkey-anti-rabbit antibody HRP-conjugated donkey-anti-rabbit antibody
• Example 20 Nanobody binding on living cells overexpressing plgR In order to determine binding specificity to plgR in cells Nanobody 4B 1 1 was tested in an immunofluorescence setup (adapted from Klapisz 2002).
• Example 21 Nanobodies are able to bind the hpIgR in its native form
• Nanobodies are able to bind the hpIgR in its native form
• an immunoprecipitation experiment was performed, nanobodies, containing a His-tag, were allowed to bind to hpIgR in cell Iy sates and fished out with talon beads.
• the hpIgR was detected on blot with a-hSC and DAG-PO.
• As a control the VHHs bound to the beads were detected with a-myc and DAM-PO.
• the result of this immunoprecipitation experiment clearly shows that the VHHs: 1D2, 4Bl 1, 4B7 and 1D7 are able to bind to hpIgR in cell lysates.
• the receptor could be detected in the four lanes containing Iy sates with the hpIgR binding VHHs.
• Empty talon beads and nanobodies directed against the EGF receptor were not able to detect the receptor and the receptor was also not detected in lysates of untransfected MDCK cells.
• the lysatc 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 In order to check the transcytosis capacity of the Nanobodies, transwell experiments were performed. Two different nanobodies, namely 1D2 (IgA-competing) and 4B7 (non- competing) were recloned into a phagemid vector and monoclonal phages were produced. We wanted to show that the phages were able to transcytose across MDCK cells expressing plgR from the basolateral to the apical side.
• 1D2 IgA-competing
• 4B7 non- competing
• the transcytosis assay is performed with fully polarized MDCK cells, seeded on 1 cm , 0.4 ⁇ m collagen- coated PTFE transwell filters (Costar). Lucifer Yellow (LY) was added to the basolateral chamber one hour before the experiment as a control for monolayer integrity and aspecific transport. The concentration of LY in the apical chamber was determined by measuring fluorescence. When the apical LY samples showed no leakage or a-specific transport transcytosis experiments are performed. 10 6 phages were added to the basolateral chamber of the Transweil-system and allowed to transcytose for 5 hours. Samples were taken from the apical chamber and the total amount of transcytosed phages was determined.
• Monoclonal phages 1D2 and 4B7 are able to transcytose across the monolayer of MDCK cells bearing the hpIgR, whereas they can not cross the MDCK cells without hpIgR. Also an irrelevant phage expressing GST-binding nanobody did not transcytose across transfected or untransfected cells.
• 2 llamas (117, 1 18) 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.
• PBLs peripheral blood lymphocytes
• RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage is prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored at 4 0 C for further use.
• Example 25 Selecting repertoires for binding: to serum albumin.
• human serum albumin (Sigma A-8763) was coated onto Maxisorp 96- well plates (Nunc, 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 triethanolamine (TEA) and neutralized with IM Tris-HCl pH 7.5.
• TAA triethanolamine
• phage libraries were incubated with antigen at physiological pH and eluted at acidic pH as follows.
• human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 ⁇ g/ml overnight (ON) at room temperature (RT). Plates are blocked with 4% Marvel in PBS pH 7.3 for 2h at RT. After 5 washes with PBS/0.05% Tween20 (PBST) pH 7.3, phages were added in 2% Marvel/PBS pH 7.3 and incubated for 2h at RT.
• PBST PBS/0.05% Tween20
• 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 mM sodium acetate + 115 mM NaCl) adjusted to pH 7.3. Unbound phages were removed by 10 washes with CPA/0.05%Tween20 (CPAT) ⁇ H7.3 ; 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.
• 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 was 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 PBSpH 7.3. Bound phage was eluted with lmg/ml trypsin/CPA pH 7.3 for 30 min at RT.
• Example 27 Library evaluation by ELISA.
• Periplasmic extracts of individual Nanobodies were screened for albumin specificity by ELISA on solid phase coated human serum albumin. Detection of Nanobody fragments bound to immobilized human serum albumin was carried out using a biotinylated mouse anti- his antibody (Serotec MCAl 396B) detected with Streptavidin-HRP (DakoCytomation #P0397). The signal was developed by adding TMB substrate solution (Pierce 34021) and detected at a wavelength of 450 ran. A high hit rate of positive clones can already be obtained after panning round 1.
• Example 28 Selection for conditional or pH-sensitive binding of Nanobodies to albumin by ELISA.
• phage libraries may be incubated with antigen at physiological pH and eluted at acidic pH as follows.
• human serum albumin (Sigma A-8763) is coated onto Maxisorp
• 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
• 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 PBSpH 7.3. Bound phage was eluted with lmg/ml trypsin/CPA pH 7.3 for 30 min at RT. In all selections, enrichment is observed. The output from each selection was re-cloned as a pool e.g. into the expression vector pAX51. Colonies are picked and grown in 96 deep-well plates (1ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ⁇ 80 ⁇ l) are prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
• Example 29 Screening of Nanobody repertoire for the occurrence of a pH-sensitave interaction via surface plasmoa resonance (BIAcore).
• Human serum albumin was immobilized on a CM5 sensor chip surface via amine coupling using NHS/EDC for activation and ethanolamine for deactivation (Biacore amine coupling kit)
• Nanobodies to albumin (Biacore) are as follows: 1OmM Sodium citrate (NajC ⁇ HsCh) + 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 pH1.5 + 0.1% P20. Evaluation was done using Biacore TlOO evaluation software.
• the off rate of different Nanobodies at pH7 and pH5 is documented in Table E-I.
• the majority of the Nanobodies (4A2, 4A ⁇ , 4B5, 4B6, 4B8, 4C3, 4C4, 4C5, 4C8. 4C9, 4D3, 4D4, 4D7 ad 4D 10 have a faster off rate at pH 5 compared with pH 7 (2-6 fold difference in off rate).
• the Nanobody 4A9 has a slower off-rate at pH 5 compared to pH 7 (0.54 fold difference in off rate).
• binding to antigen does not change at different pH. Direct screening of nanobody repertoires for conditional binding to antigen can thus be used.
• Example 30 Screening for conditional binding of Naaobodies 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 tiler 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).
• 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.
• Table E-2 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 E- 3 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.
• Table E-2 Nanobodies (Clones) that oi ⁇ ly bind in neutral conditions but not in acidic condition
• Example 31 Analysis of effect of conditional binding on pharmacokinetic behavioiir of the Polypeptide of the Invention. a) Construction of bi- or multispecifie naraobody format
• Bispecific nanobodies are e.g. generated by construction of a C-terminal pH dependent FcRn binding Nanobody, a 9 amino acid Gly/Ser linker (e.g. GGGGSGGGS) and an N-terminal anti-target Nanobody, e.g. an N-terminal Polypeptide with 2 Nanobodies against EPO-R functioning as agonist on human or murine EPO-R or an N-terminal Polypeptide with 2 Nanobodies against EPO-R functioning as agonist on human or murine GHR.
• These constructs may be expressed in E.coli as c-myc, His6-tagged proteins and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromotagraphy (SEC).
• IMAC immobilized metal affinity chromatography
• SEC size exclusion chromotagraphy
• conditional pH- binding properties of the anti-FcRn or plgR Nanobody or dAbs within the muMspecific nanobody formats e.g. are evaluated via surface plasmon resonance (BIAcore), e.g. a conditional binder as disclosed in this application is linked to one or more nanobody or dAbs binding to one or more protein target(s), e.g. is linked to 2 Nanobodies directed against Epo-R or HGR.
• Cross-reactivity to cynomolgus serum albumin is also assessed.
• Human and cynomolgus FcRn or plgR are immobilized on a CM5 sensor chip surface via amine coupling using NHS/EDC for activation and ethanolamine for deactivation (Biacore amine coupling kit)
• Nanobodies to FcRn or plgR are as follows: 1OmM Sodium citrate (NasC ⁇ HsO ⁇ ) + 1OmM 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 pHl .5 + 0.1% P20. Evaluation is done using Biacore TlOO evaluation software.
• Example 32 Pharmacokinetic profile of multispecific nanobody formats in eynomolgus monkey delivered by i.v, injection
• a pharmacokinetic study is conducted in cynomolgus monkeys.
• a Polypeptide of the Invention e.g. Epo-R or HGR agonstic bivalent polypeptide with FcRn or plgR pH dependent binding block, i.e. 2 Epo-R or 2 HGR 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.
• concentration in the plasma samples is determined as follows: Maxisorb micro titer plates (Nunc, Article No. 430341) are coated overnight at 4°C with 100 ⁇ l of a 5 ⁇ g/ml solution of the Polypeptide of the Invention in bicarbonate buffer (50 mM, pH 9.6).
• Plasma samples and serial dilutions of polypeptide-standards are diluted in PBS in a separate non-coated plate (Nunc, Article No. 249944) to obtain the desired concentration/dilution in a final sample matrix consisting of 10% pooled cynomolgus plasma in PBS. All pre-dilutions are incubated for 30 minutes at RT in the non-coated plate.
• the coated plates are washed three times (PBS containing 0.1% Tween20), and an aliquot of each sample dilution (lOO ⁇ l) is transferred to the coated plates and allowed to bind for 1 hour at RT. After sample incubation, the plates are washed three times (PBS containing 0.1% Tween20) and incubated for 1 hour at RT with 100 ⁇ l of a 100 ng/ml solution of sIL6R in PBS (Peprotech, Article No. 20006R).
• Each individual plasma sample is analyzed in two independent assays and an average plasma concentration is calculated for pharmacokinetic data analysis, All parameters are calculated with two-compartmental modeling, with elimination from the central compartment.
• Example 33 Preparation of various pharmaceutical orally deliverable compositions a) Capsules comprising the Polypeptides 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 enteric coating material preferably may be one which will provide for release of polypeptide at about pH 6.0-6.5 such as, for example, CAP and HPMC-AS.
• the enterocoating is carried out by methods known per se in the art, e.g., according to Remington Pharmaceutical Sciences, p. 1614-1615 (1975, 15th Ed., Mack Pub. Co.) and Theory and Practice of Industrial Pharmacy, Lackman, Liberman & Caning, p. 116-117, 373- 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 Polypeptides of the Invention e.g. the herein described examples, e.g. agonistic HGR or EpoR polypeptides (i.e. bispecific construct comprising 2 Nanobodies against HGR 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 HGR or EpoR polypeptides i.e. bispecific construct comprising 2 Nanobodies against HGR 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)
• a liquid formulation will generally consist of a solution or suspension containing the biologically active polypeptide, e.g. the Polypeptide 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 biologically active polypeptide e.g. the Polypeptide 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 Polypeptide 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-mi scible solvents such as, for example, ethanol, glycerin, propylene gylcol and sorbitol, or mixtures of any of the foregoing.
• a pharmaceutically acceptable liquid carrier such as, for example, a sterile aqueous carrier or water-mi scible solvents such as, for example, ethanol, glycerin, propylene gylcol and sorbitol, or mixtures of any of the foregoing.
• Example 34 In vivo model to test systemic delivery In Vivo Model for Epo-R agonist read out fSpiekermann et al, 2002, supra).
• mice 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. Isoflurane by inhalation and the different Polypeptides of the Invention (e.g. as disclosed above, e.g. construct comprising 2 anti-mouse non-neutralizing Epo-R Nanobodies (e.g. with 9 GIy linker), optionally comprising a pH independent or pH dependent anti-mouse FcRn or plgR Nanobody (i.e.
• binding at gut pH, about pH 6, but released at blood pH7 or more are injected intraperitoneally, gauged into small intestine, fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart), or administered orally by a enterically coated capsule for mouse consumption, e.g. capsule from example 32.
• Mice are killed by CO 2 inhalation 8 h or 4 d later and whole blood is obtained by cardiac puncture.
• the agonistically acting Nanobody construct may have a Koff equal or lower than 1 nM since interaction of the first site of Epo for EpoR is of high affinity (dissociation constant 1 nM) while the second binding interaction is much weaker (1 uM). To be determined e.g. in BioCore experiments.
• In vivo model for HGR agonist read out -In vivo assay to test GHR agonists (Wang et al, 1996 Molecular and Cellular Endocrinology, Volume 116, Issue 2, 5 February 1996, Pages 223-226) -Studies on promotion of animal growth using GH deficient hypophysectomized rats.
• mice are anaesthetized with e.g. Isoflurane by inhalation and the different Polypeptides 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 agonistically acting Nanobody construct against GHR from above may have a Koff equal or lower than 0.3 nM since interaction of GH to GHR-dimer was reported to be in the range of 0.3 nM (Cunningham et al, 1989, Science 244:1081-1085.). To be determined e.g. in BioCore experiments. Preferred embodiments:
• a pharmaceutical composition for oral administration comprising a therapeutically effective amount of a polypeptide comprising one or more single variable domain(s) and a pharmaceutically acceptable enteric coating.
• composition according to embodiment 1, wherein said polypeptide comprises or essentially consists of a single Nanobody, domain antibody, single domain antibody or "dAb", preferably a Nanobody.
• composition according to embodiment 1, wherein said polypeptide comprises or essentially consists of at least two Nanobodies, domain antibodies, single domain antibodies or "dAbs", preferably a Nanobody.
• composition according to embodiment 3, wherein said polypeptide 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 embodiment 1 wherein said polypeptide 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 embodiment 5, wherein said one or more amino acid sequence is a Nanobody, a domain antibody, a single domain antibody or a "dAb", preferably a Nanobody.
• composition according to embodiment 6 wherein said one or more amino acid sequence is directed against a serum protein.
• 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 embodiment 8, wherein said one or more amino acid sequences is a Nanobody, a domain antibody, a single domain antibody or a "dAb" , preferably a Nanobody.
• composition according to embodiment 1, wherein said polypeptide 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 embodiment 11, wherein said Nanobody, domain antibody, single domain antibody or "dAb” is a humanized V HH or a camelized VH-
• composition according to embodiment 20 wherein, upon oral administration of said composition to a subject, the polypeptide reaches a Cmax in the gut of at least 1% of the Cmax that is reached following parenteral administration of the same amount of polypeptide.
• composition of any of embodiments 1 to 24, wherein the enteric coating is an anionic polymer comprising methacrylic acid and methacrylates and dissolve at ranges from pH 5.5. to pH 7.
• a permeability enhancer such as e.g. acylcarnitine or N-(5-chlorosalicyloyl)-8-aminocaprylic acid.
• polypeptide comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of plgR.
• composition of embodiment 31, wherein the polypeptide comprises a) at least one Nanobody directed against a target molecule; and b) a Nanobody directed against the extracellular part of 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 10 nM.
• composition of embodiment 32, wherein the polypeptide 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 embodiment 33, wherein the polypeptide 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 embodiment 31, wherein the polypeptide 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 6.
• composition of embodiment 31, wherein the polypeptide 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 cross-blocks FcRn Nanobody of SEQ ID NOs: 1 to 6.
• the polypeptide 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 has 70% ; 75%, 80%, 85%, 90% sequence identity (measured e.g. with blast
• composition of embodiment 32, wherein the polypeptide 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: 7 to 27.
• composition of embodiment 30, wherein the polypeptide 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: 7 to 27.
• composition of embodiment 3O 5 wherein the polypeptide 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: 7 to 27.
• composition of any of embodiments 31 to 42, wherein the binding of the Nanobody directed against the extracellular part of plgR, FcRn or Vit B 12 receptor is pH dependent.
• composition of embodiment 43 wherein the the Nanobody directed against the extracellular part of plgR, FcRn or Vit Bl 2 receptor binds at pH6 or lower, e.g. pH5, to its receptor and does significantly less (e.g. 2, 3, 4, 5, or 10 times or not at all) bind to its receptor at pH7 and higher.
• composition of any of embodiments 31 to 44, wherein the polypeptide has antagonistic properties to the target molecule is provided.
• composition of embodiment 45 wherein the polypeptide is an agonist to Epo-R.
• composition of embodiment 49 wherein the proteolytically stabilized properties are resulting from polypeptides consisting essentially of proteolytically stabilized (i.e. screened for proteolytically stabilzed) Nanobodies, e.g. if consisting of more than one Nanobodies the Nanobodies are e.g. linked with Gly/Ser linkers.
• a method for delivering a polypeptide comprising or essentially consisting of a Nanobody. a domain antibody, a single domain antibody or "dAb" to the bloodstream of a subject comprising the step of orally administering a composition according to any of embodiments 1 to 50 to said subject.
• a method for delivering a polypeptide comprising or essentially consisting of a Nanobody, a domain antibody, a single domain antibody or "dAb" to the gut of a subject comprising the step of orally administering a composition according to any of embodiments 1 to 50 to said subject.
• a method for the prevention and/or treatment of a subject in need of polypeptide comprising or essentially consisting of a Nanobody, a domain antibody, a single domain antibody or a ''dAb' " , said method comprising the step of orally administering to said subject a Nanobody, a domain antibody, a single domain antibody or a "dAb" 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 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 imrmmo therapy comprising oral administering to a subject suffering from or at risk of a diseases and/or disorders that can be cured or alleviated by immunotherapy with a Nanobody, a domain antibody, a single domain antibody or a "dAb", a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or u dAb" and/or of a composition comprising the same.
• Nanobody, a domain antibody, a single domain antibody or a "'dAb” for the preparation of a composition according to any of embodiments 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”.
• composition according to any of embodiments 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
• a method for the prevention and/or treatment of a subject in need of a Nanobody, a domain antibody, a single domain antibody or a '"dAb" that is directed against a target in the kidney or bladder 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 kidney or bladder comprising orally administering to said subject a therapeutically effective amount of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in the kidney or the bladder and/or of a composition comprising the same.
• Nanobody Use of a Nanobody, a domain antibody, a single domain antibody or a "dAb" directed against a target in the kidney or the bladder for the preparation of a composition according to any of embodiments 1 to 27 for the prevention and/or treatment of at least one a disease or disorder of the kidney or bladder.
• a method for the prevention and/or treatment of a subject in need of a Nanobody, a domain antibody, a single domain antibody or a "dAb” that is directed against a target in the lung 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 lung comprising orally administering to said subject a therapeutically effective amount of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target in the lung and/or of a composition comprising the same.
• Nanobody a domain antibody, a single domain antibody or a "dAb" directed against a target in the lung for the preparation of a composition according to any of embodiments 1 to 50 for the prevention and/or treatment of at least one disease or disorder of the lung.
• a method for the prevention and/or treatment of a subject in need of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target on a tumor cell comprising orally administering, to said subject a therapeutically effective amount of said Nanobody, domain antibody, single domain antibody or £i dAb" 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 Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against a target on a tumor and/or of a composition comprising the same.
• Nanobody a domain antibody, a single domain antibody or a "dAb" directed against a target on a tumor for the preparation of a composition according to any of embodiments 1 to 50 for the prevention and/or treatment of at least one a tumor related disease or disorder.
• a method for the prevention and/or treatment of a subject in need of a Nanobody, a domain antibody, a single domain antibody or a "dAb” that is directed against a target in gut 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 Nanobody, a domain antibody, a single domain antibody or a "dAb” 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 method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Nanobody, a domain antibody, a single domain antibody or a "dAb” that is directed against TNF said method comprising orally administering to said subject a therapeutically effective amount of said Nanobody. domain antibody, single domain antibody or "dAb” and/or of a composition comprising the same.
• a disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease)
• a therapeutically effective amount of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is directed against TNF and/or of a composition comprising the same.
• Nanobody a domain antibody, a single domain antibody or a "dAb" directed against TNF for the preparation of a composition according to any of embodiments 1 to 50 for the prevention and/or treatment of at least one disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease).
• an autoimmune disease such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease
• 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 Nanobody, a domain antibody, a single domain antibody or a "dAb” that is directed against vWF 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 related to platelet- mediated aggregation (such as e.g. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis
• Nanobody a domain antibody, a single domain antibody or a "dAb" directed against vWF for the preparation of a composition according to any of embodiments 1 to 50 for the prevention and/or treatment of at least one disease or disorder related to platelet-mediated aggregation (such as e.g.
• 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-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).
• a method for the prevention and/or treatment of a subject in need of a Nanobody, a domain antibody, a single domain antibody or a "dAb' " 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 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 at least one disease or disorder that can be prevented and/or treated by administering to a subject suffering from said disease or disorder a Nanobody, a domain antibody, a single domain antibody or a "dAb” that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex, said method comprising orally administering to said subject a therapeutically effective amount of said 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. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomeruloriep3 ⁇ ritis, 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, Castêtan's disease, IgM gamma
• Nanobody a domain antibody, a single domain antibody or a "dAb" directed against IL-6, IL-6R and/or IL-6/IL-6R complex for the preparation of a composition according to any of embodiments 1 to 27 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).
• 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).
• a method for the prevention and/or treatment of an acute disorder or disease comprising orally administering to said subject a therapeutically effective amount of a Nanobody, a domain antibody, a single domain antibody or a "dAb" that is capable of alleviating the symptoms of or curing said disorder or disease.
• Nanobodies, domain antibodies, single domain antibodies or “dAbs” directed against an epithelial trans-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 administering to a subject a Nanobody, a domain antibody, a single domain antibody or a "dAb” or a composition comprising the same and detecting said Nanobody, domain antibody, single domain antibody or "dAb”.

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