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.
Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.
Administration of conventional low molecular weight drugs by non-invasive routes has been a well established practice. Therapeutic peptides and proteins, however, are often unstable, have large molecular weights and are polar in nature. These properties lead to poor permeability through biological membranes. When administered orally, they are susceptible 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.
However, injection, infusion and oral administration are significantly less convenient than, and involve more patient discomfort than, oral administration. Often this inconvenience or discomfort results in substantial patient non-compliance with the treatment regimen. Thus, there is a need in the art for more effective and reproducible oral administration of polypeptides like e.g. single variable domains and/or construct thereof.
Proteolytic enzymes of both the stomach and intestines may degrade polypeptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of polypeptides that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas (typically having neutral to basic pH optima). Specific difficulties arising from the oral 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 basolateral 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.
First, peptides and proteins are expensive to manufacture either by chemical synthesis or recombinant DNA technologies. Therefore, the more one increases bioavailability, the lesser the amounts that will be required in an oral formulation of a therapeutic drug (economic issue).
Second, the greater the bioavailability of an oral peptide, the less the variability in the dosage absorbed by an individual on a day to day basis (safety issue).
Third, the greater the bioavailability of an oral peptide, the less the concern about breakdown products of the peptide since such breakdown products can act as agonists or antagonists of the receptors where the peptide binds to elicit biological activity (safety issue).
Accordingly delivery of therapeutic polypeptides through the oral route receives great attention; it has not been successful and is considered a big hurdle to biological drugs. The main reasons are intrinsic poor permeability of intestinal wall and fast proteolytic degradation in stomach and gut. There is as of today no oral delivery of larger polypeptides (of 100 amino acids and more) approved for human use and there is no established procedure or know how in the art how to formulate a polypeptide for oral to gut-local or systemic delivery, in particular to systemic delivery.
The present inventors have now found that a certain class of therapeutic polypeptides, 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 biologics, 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.
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. 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.
In one of the embodiments of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by 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).
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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. In a preferred embodiment, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, 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, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, 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, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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. In a preferred embodiment, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, 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, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, 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, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder; and [d) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or e) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcarnitine and/or Eligen® carrier technology].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, e.g. Eudragit L30D-55 (Roehm Pharma Polymers); and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art, 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. 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”. In a preferred embodiment, 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”. In a further preferred embodiment, 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). In a further embodiment, the Nanobody, domain antibody, single domain antibody or “dAb” is derived from a VH or VHH. 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 “FR's” and “complementarity determining regions” or “CDR's”.
It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the Polypeptides of the Invention, and/or to use Polypeptides of the Invention comprising or essentially consisting of the same, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles, and derivatives will be described in the further description herein.
According to a specific, but non-limiting embodiment, the amino acid sequences of the Nanobodies and/or Polypeptides of the Invention can be “humanized”, “camelized” or modified as further described herein.
Generally, 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 “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”.
According to one specific, but non-limiting embodiment, 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”. As further described herein, 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. According to another specific, but non-limiting embodiment, 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.
According to yet another specific, but non-limiting embodiment, 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. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies, domain antibodies, single domain antibodies or “dAbs”. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein. In a specific embodiment, 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. In another specific embodiment, 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.
In the above constructs, 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. Some suitable but non-limiting examples of such linkers will become clear from the further description herein. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the 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).
Preferably, 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). Examples are a 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.
In one preferred embodiment of the invention, 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 allow the resulting Polypeptide of the Invention to be cross the intestine wall of the gut. In particular, 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 macromolecules 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. Furthermore, 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.
In another preferred embodiment, 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). In particular, 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. Examples of suitable Nanobodies against mouse or human serum albumin are described in the applications WO 03/035694, WO 04/041865 and WO 06/122825.
In yet another preferred embodiment, 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. the kidney, bladder, lung and/or brain) (optionally linked via one or more suitable linker sequences). Again, 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).
The 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, pIgR 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.
Thus, in a further aspect, 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. The use of such additional excipients is well known to those skilled in the art of pharmacology. Some non-limiting examples of such additional excipients are found in e.g. Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy) 21st edition.
Optionally, the composition of the invention also comprises other additives and/or agents. Accordingly, in another embodiment of the invention, a composition is provided 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. The use of additives such as preservatives, buffering agents, antioxidants, bulking agents and/or viscosity builders are known to those skilled in the art of pharmacology and are also further described e.g. Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy) 21st edition.
In various embodiments, 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.
In another embodiment of the invention, the composition may additionally comprise one or more further therapeutic ingredient (or active substances). These combinations of therapeutic ingredients and/or active substances (e.g. also including constructs covalently linking the Polypeptides of the Invention) will also become clear from the further description herein.
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.
The 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. In an embodiment of the invention, 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. In another embodiment, 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. In a further embodiment of the invention, 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. In another further embodiment, 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. In another further embodiment, 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. In an embodiment of the invention, 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. In another embodiment of the invention, 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 Nanobody. In another further embodiment of the invention, 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.
In yet another aspect, 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. In an embodiment of the invention, the bioavailability for the Polypeptide of the Invention comprising at least a Nanobody and/or dAbs, more preferably a 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.
The above and other aspects, embodiments and advantages of the invention will become clear from the further description herein below.
(1)Sometimes also considered to be a polar uncharged amino acid.
(2)Sometimes also considered to be a nonpolar uncharged amino acid.
(3)As will be clear to the skilled person, the fact that an amino acid residue is referred to in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and/or at a pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged and/or uncharged at such a higher or lower pH, as will be clear to the skilled person.
(4)As is known in the art, the charge of a His residue is greatly dependant upon even small shifts in pH, but a His residu can generally be considered essentially uncharged at a pH of about 6.5.
Without being limited thereto, 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. In accordance with the terminology used in the references below, the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH 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 “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “VL domains”).
Thus—without being limited thereto—the polypeptide or protein of the invention has an amino acid sequence that comprises or essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 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.
a) Inhibition of Proteolytic Activity that Degrades Polypeptides in Stomach and Gut
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. Such agents are generally known to the skilled person in the art and may be found in e.g. Remington, supra. An example of protease inhibitor is an organic acid such as citric or acetic acid. Protease inhibitors are readily available for the skilled person in the art.
b) Development of Protease-Resistant Polypeptide Analogs that Retain Biological Activity
Hermsen et al. (see Harmsen M M, van Solt C B, van Zijderveld-van Bemmel A M, Niewold T A, van Zijderveld F G. Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol. 2006 Feb. 1; 1-8) showed that stringent selection for proteolytic stability resulted in seven Nanobodies (or VHHs) with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling they further obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These Nanobodies or VHHs differed by at most ten amino acid residues from each other and were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Similarly, the invention provides pharmaceutical compositions comprising proteolytically stable single variable domains, e.g. Nanobodies or VHHs, wherein said proteolytically stable Nanobodies can be formatted into bi- and/or multivalent (and multimeric) constructs, e.g. into the constructs, polypeptides of the invention.
It is a further embodiment of the invention to provide 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. As mentioned herein, the single variable domains, e.g. Nanobodies and/or dAbs and constructs described herein may be pegylated, or contain one or more (additional) amino acid residues that allow for pegylation and/or facilitate pegylation. Two preferred, but non-limiting examples of such polypeptides are TNF55 and TNF56 as described in WO/2006/122786, which both contain an additional cysteine residue for easy attachment of a PEG-group.
d) Protection of the Polypeptide from Proteolytic Degradation by e.g. Enteric Coating
Any enteric coating that protects the peptide from stomach proteases and which releases active components of the invention in the intestine is suitable. The enteric coating functions by providing a coating that does not dissolve in low pH environments, such as the stomach. Many enteric coatings are known in the art, and are useful in accordance with the invention. Examples include cellulose acetate phthalate, hydroxypropylmethylethylcellulose succinate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, and methacrylic acid-methyl methacrylate copolymer. It is very desirable that all of the active components be released from the dosage form, and solubilized in the intestinal environment as simultaneously as possible. It is preferred that the dosage form release the active components in the small intestine.
e) Improvement of Active (e.g. Receptor Mediated or M-Cell Mediated) Trans-Epithelial Transport of the Polypeptides
It is also known that Fc receptors are involved in transcytosis recycling of proteins and other (biological) molecules. For example, pIgR, FcRn, and Vit B12 receptor is known to be involved in transcytosis through biological membranes such as epithelial layers, e.g. in adult human gut, and FcRn is known to be involved in the recycling of albumin and IgG (see for example Chaudhury et al., The Journal of Experimental Medicine, vol. 3, no. 197, 315-322 (2003)). Thus, the invention provides building blocks, i.e. single variable domains such as Nanobodies and/or dAbs binding to pIgR, FcRn and/or the Vit B 12 receptor. Furthermore, the building block may also be the natural ligand or fragment of ligand, i.e. human Fc part. It is an embodiment of the invention to provide 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. FcRn, Vit B12 or pIgR, preferably FcRn or pIgR, more preferably FcRn, binding Nanobody). 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 (naïve 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 trans-membrane protein or cell lines transfected with an epithelial trans-membrane protein gene to allow the easy selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs binding to the epithelial trans-membrane protein. This avoids the need for protein expression and purification, speeding up significantly the generation of membrane crossing Nanobodies, domain antibodies, single domain antibodies or dAbs.
In another embodiment, the invention includes a selection method using cells to allow the selection of phage single variable domains, Nanobodies, domain antibodies, single domain antibodies or dAbs that show receptor mediated internalization. Said method comprises adding the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to the cells and recovering the phage Nanobodies, domain antibodies, single domain antibodies or dAbs from the cells that have undergone internalization. In yet another embodiment, the invention includes a selection method using cells seeded on a filter or in a Transwell system or Boyden chamber to allow the selection of phage Nanobodies, domain antibodies, single domain antibodies or dAbs that transcytose through the cell monolayer. Said method comprises adding the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to compartment 1, allow the phage Nanobodies, domain antibodies, single domain antibodies or dAbs to migrate across the cell monolayer and harvest the phage Nanobodies that migrate in compartment 2. Alternatively, the Polypeptides of the Invention comprising e.g. at least a Nanobody or a dAb against a Target Molecule, may also be suitably formulated per se for oral delivery e.g. in the form of a powder (such as a freeze-dried or micronized powder) or mist.
In an embodiment, the Polypeptides of the Invention comprising e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, may also form a sequence or signal that allows said Polypeptides of the Invention comprising e.g. at least one Nanobody and/or dAbs, preferably a Nanobody, to be directed towards and/or to penetrate or enter into specific gut epithelial cells, or parts or compartments of said cells, and/or that allows the Polypeptides of the Invention comprising e.g. at least one Nanobody and/or dAb, preferably a Nanobody, to penetrate or cross a biological barrier such as the gut wall or membrane.
In another preferred embodiment, 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.
Examples of such Nanobodies, domain antibodies, single domain antibodies or dAbs include Nanobodies, domain antibodies, single domain antibodies or dAbs that are directed towards specific cell-surface proteins, receptors, markers or epitopes of the gut membrane cells.
In this context, the 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.
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.
In one embodiment of the present invention, 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, pIgR and/or VitB12 receptor as disclosed in the experimental part.
In another embodiment, 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. at least one Nanobody and/or a dAb, preferably a Nanobody, for crossing or passage through the epithelial membrane. Examples of such a therapeutic polypeptide or agent are Nanobodies against FcRn, pIgR and/or VitB12 receptor.
In yet another embodiment, 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. The resulting Polypeptide, upon binding of the ligand to the epithelial trans-membrane protein, is transported through the membrane. An example of such ligand (e.g. a natural ligand) of the epithelial trans-membrane protein is the Fc unit or fragment thereof of a human antibody, e.g. the Fc unit of human IgG1.
In yet another embodiment of the present invention, 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. pH5 or pH6, than at neutral physiological pH such as pH 7 or more, e.g. pH 7.4. Such pH dependent single variable domains, e.g. Nanobodies, are exemplified in this application (pH dependent human FcRn and pH dependent human serum albumin binders) and are disclosed in the experimental part.
In yet another embodiment, 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. pH5 or pH6, than at neutral physiological pH such as pH 7 or more, e.g. pH 7.4. The resulting Polypeptide, upon binding of the ligand to the epithelial trans-membrane protein, is transported through the membrane. An example of such ligand (e.g. a natural ligand) of the epithelial trans-membrane protein is the Fc unit or fragment thereof of a human antibody, e.g. the Fc unit of human IgG1.
f) Increase of Half-Life of the Polypeptide of the Invention in Human Body, e.g. at Target Site, for e.g. Those Active Polypeptides that Require a Sustained Presence for Therapeutic Efficacy by Addition of Suitable Excipient, e.g. Biodegradable Polymer, and/or by Covalently Binding an Unit Allowing for Longer Half Life
In one specific aspect of the invention, 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 Invention. Examples of 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, “dAb”'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrine; reference is made to the further description and references mentioned herein, see e.g. also WO 2007/112940); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N.V. entitled “Peptides capable of binding to serum proteins” of Ablynx N.V. filed on Dec. 5, 2006 (see also PCT/EP2007/063348). Generally, 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. For example, 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. In a preferred, but non-limiting aspect of the invention, 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. In another preferred, but non-limiting aspect of the invention, 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. For example, 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).
The Compositions of the Invention may further comprise one or more permeation enhancer. As used herein, trans-epithelial permeation enhancers include agents which enhance the release or solubility (e.g., from a formulation delivery vehicle), diffusion rate, penetration capacity and timing, uptake, residence time, stability, effective half-life, peak or sustained concentration levels, clearance and other desired delivery characteristics (e.g. as measured at the site of delivery, or at a selected target site of activity such as the bloodstream and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder, lung and/or brain) of the Polypeptides of the Invention or of additional biologically active ingredient(s). Enhancement of passive transport through intestinal gut wall can thus occur by any of a variety of relevant mechanisms, for example by increasing the diffusion, increasing membrane fluidity, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing mucosal membrane components (e.g. lipids), changing non-protein and protein sulfhydryl levels in epithelial tissues, increasing water flux across the surface, modulating epithelial junctional physiology, reducing the viscosity of mucus overlying the epithelium, reducing mucociliary clearance rates, increasing blood flow and other mechanisms. Suitable permeability enhancing agents will be clear to a person skilled in the art of pharmacology and are further described hereafter. Such agents may be used in suitable amounts known per se, which will be clear to the skilled person based on the disclosure and prior art cited herein.
Permeability enhancing agents include (a) aggregation inhibitory agents, (b) charge modifying agents, (c) mucolytic or mucus clearing agents, (d) ciliostatic agents; (f) membrane penetration-enhancing agents such as acylcarnitine, (g) modulatory agents of epithelial junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (h) vasodilator agents, and (i) stabilizing delivery vehicles, carriers, supports or complex-forming species with which the Polypeptide of the Invention is effectively combined, associated, contained, encapsulated or bound to stabilize the Polypeptide of the Invention for enhanced intestinal transport. These agents are further exemplified—without being limiting as additional agents comprised in the compositions of the present invention—in WO98034632C2, WO98034632, WO9834632, WO9834632, WO9736480 and/or WO9630036.
In a further embodiment, a membrane penetration-enhancing agent is added to the composition of the present invention. Different membrane penetration-enhancing agents have been described such as (i) a surfactant, (ii) a bile salt or bile salt derivative, (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an 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-acetylamino acid or salt thereof, (xvi) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis, (xix) cationic polymers, or (xx) any combination of the membrane penetration enhancing agents of ((i)-(xix)). The membrane penetration-enhancing agent can be selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Alternatively, long-chain amphipathic molecules, for example, deacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and the bile salts (e.g., unsaturated cyclic ureas and Transcutol), may be employed to enhance mucosal penetration of the Nanobodies, polypeptides or proteins of the invention. In additional aspects, surfactants (e.g., Tween 80, Poloxamer 188, polysorbates; further non-limiting examples of surfactants are also provided in EP 490806, U.S. Pat. No. 5,759,565, and WO 04/093917) are employed as adjunct compounds, processing agents, or formulation additives to enhance oral delivery of the Nanobodies, polypeptides or proteins of the invention. These penetration-enhancing agents typically interact at either the polar head groups or the hydrophilic tail regions of molecules that comprise the lipid bilayer of epithelial cells lining the oralmucosa (Barry, Pharmacology of the Skin, Vol. 1, pp. 121-137, Shroot et al., Eds., Karger, Basel, 1987; and Barry, J. Controlled Release 1987; 6: 85-97). Interaction at these sites may have the effect of disrupting the packing of the lipid molecules, increasing the fluidity of the bilayer, and facilitating transport of the Polypeptides of the Invention across the mucosal barrier. Additional non-limiting examples of membrane penetration-enhancing agent are described in WO 04/093917, WO 05/120551 and Davis and Illum (Clin. Pharmacokinet 2003, 42: 1107-1128).
In various embodiments of the invention, 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.
While the mechanism of absorption promotion may vary with different permeability-enhancing agents of the invention, useful reagents in this context will not substantially adversely affect the tissue and will be selected according to the physicochemical characteristics of the particular Polypeptide of the Invention or other active ingredients or delivery enhancing agent. In this context, delivery-enhancing agents that increase penetration or permeability of the gut wall will often result in some alteration of the protective permeability barrier of the gut. For such delivery-enhancing agents to be of value within the invention, it is generally desired that any significant changes in permeability of the gut be reversible within a time frame appropriate to the desired duration of drug delivery. Furthermore, there should be no substantial, cumulative toxicity, nor any permanent deleterious changes induced in the barrier properties of the gut with long term use.
Some preferred embodiments are using the above disclosed strategies are provided below:
In one of the embodiments of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, more preferably agonistic polypeptides, systemic and/or local (i.e. topical gut) delivery, is provided through oral administration by protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of a suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder. In a preferred embodiment, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled, person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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. In a preferred embodiment, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) develop protease-resistant polypeptide analogs that retain biological activity, e.g. pharmaceutical oral compositions comprising Target Molecule binding single variable domains, e.g. Nanobodies or dAbs, selected for protease resistance by at least 2, 3, 4, 5 10, 20, 50 100 folds (see e.g. experimental part); c) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH5 or pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and d) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder. In a preferred embodiment, the unit extending half-life is also able to improve active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. a FcRn binding unit is able to prolong half/life and improve active receptor mediated trans-epithelial transport in the gut.
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration provided by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, wherein said receptor binding is a high affinity binding (e.g. dissociation constant of 100 nM, preferably 10 nM, more preferably 1 nM or 100 pM, most preferred 10 pM, at pH6 or less but has 2 times less, preferably 3, 4, 5, 10, 20, 50 or 100 times less, more preferably no binding at pH7 and more, e.g. by pH dependent pIgR, pH dependent FcRn, and/or pH dependent VitB12 receptor mediated trans-epithelial transport, preferably pIgR and/or FcRn, more preferably FcRn mediated trans-epithelial transport; and c) increasing half-life of the polypeptide in human body, e.g. at target site, for e.g. those active polypeptides that require a sustained presence for therapeutic efficacy by addition of suitable excipient, e.g. biodegradable polymer, and/or by covalently binding an unit allowing for longer half life, e.g. fused Fc fragment, albumin, albumin binder, FcRn binder, and/or serum protein binder; and [d) inhibit proteolytic activity that degrades polypeptides in stomach and gut by e.g. protease inhibitors such as e.g. organic acids; and/or e) improve passive polypeptide transport (diffusion) through the mucus and epithelial membrane by e.g. permeation enhancer such as acylcarnitine and/or Eligen® carrier technology].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) improving active (e.g. receptor mediated) trans-epithelial transport of said polypeptides, e.g. by pIgR, FcRn, and/or VitB12 receptor mediated trans-epithelial transport, preferably pIgR 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].
In another embodiment of the Polypeptides of the Invention, e.g. Nanobodies or dAbs, preferably Nanobodies, systemic and/or local (i.e. topical gut) delivery is provided through oral administration by a) protecting said polypeptides from proteolytic degradation by e.g. enteric coatings known to the skilled person in the art; and b) providing continuous local (topical in gut) delivery by bacterial system, e.g. lactic acid bacteria.
Moreover, in one embodiment, 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, diethylaminoethyl dextran, and carboxymethyl cellulose, which significantly increase the stability and reduce the solid-phase aggregation of polypeptides admixed therewith or linked thereto. In some instances, the activity or physical stability of polypeptides can also be enhanced by various additives to pharmaceutical compositions comprising the Polypeptide of the Invention. For example, additives, such as polyols (including sugars), amino acids, and various salts may be used. Certain additives, in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized polypeptides. These additives can also be used within the invention to protect the polypeptides against aggregation not only during lyophilization but also during storage in the dry state. For example, sucrose and Ficoll 70 (a polymer with sucrose units) exhibit significant protection against polypeptide aggregation during solid-phase incubation under various conditions. These additives may also enhance the stability of solid polypeptides embedded within polymer matrices. Yet additional additives, for example sucrose, stabilize polypeptides against solid-state aggregation in humid atmospheres at elevated temperatures, as may occur in certain sustained-release formulations of the invention. These additives can be incorporated into polymeric melt processes and compositions within the invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing various stabilizing additives described above. Sustained release of unaggregated polypeptides can thereby be obtained over an extended period of time. A wide non-limiting range of suitable methods and anti-aggregation agents are available for incorporation within the compositions of the invention such as disclosed in WO 05/120551, Breslow et al. (J. Am. Chem. Soc. 1996; 118: 11678-11681), Breslow et al. (PNAS USA 1997; 94: 11156-11158), Breslow et al. (Tetrahedron Lett. 1998; 2887-2890), Zutshi et al. (Curr. Opin. Chem. Biol. 1998; 2: 62-66), Daugherty et al. (J. Am. Chem. Soc. 1999; 121: 4325-4333), Zutshi et al. (J. Am. Chem. Soc. 1997; 119: 4841-4845), Ghosh et al. (Chem. Biol. 1997; 5: 439-445), Hamuro et al. (Angew. Chem. Int. Ed. Engl. 1997; 36: 2680-2683), Alberg et al., Science 1993; 262: 248-250), Tauton et al. (J. Am. Chem. Soc. 1996; 118: 10412-10422), Park et al. (J. Am. Chem. Soc. 1999; 121: 8-13), Prasanna et al. (Biochemistry 1998; 37:6883-6893), Tiley et al. (J. Am. Chem. Soc. 1997; 119: 7589-7590), Judice et al. (PNAS USA 1997; 94: 13426-13430), Fan et al. (J. Am. Chem. Soc. 1998; 120: 8893-8894), Gamboni et al. (Biochemistry 1998; 37: 12189-12194).
In another embodiment, 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, N.Y., 1988). Non-limiting examples include amastatin and bestatin (O'Hagan et al., Pharm. Res. 1990, 7: 772-776). Various classes of enzyme inhibitors are extensively described and exemplified in WO 05/120551 without being limiting for use in the composition of the present invention. Another means to inhibit degradation is pegylation with PEG molecules, preferably low molecular weight PEG molecules (e.g. 2 kDa; Lee et al., Calcif Tissue Int. 2003, 73: 545-549). Also within the scope of the present invention is the use, as enzyme inhibitor, of a Nanobody, domain antibody, single domain antibody or “dAb” directed against said enzyme. Accordingly, the invention also relates to a bispecific or multispecific Polypeptide comprising or essentially consisting of one or more Nanobodies, domain antibodies, single domain antibodies or “dAbs” directed against the desired target and one or more Nanobodies, domain antibodies, single domain antibodies or “dAbs” directed against an enzyme of the stomach and/or gut.
In addition to the Polypeptide of the Invention and, optionally, one or more additives and/or agents, the composition of the invention may further comprise one or more additional therapeutic ingredients (or active substances). These therapeutic ingredients can be any compound that elicits a desired activity or therapeutic or biological response in the subject. In a preferred embodiment, two or more Nanobodies the invention may be used in combination, i.e. as a combined treatment regimen.
As indicated above, the pharmaceutical composition of the invention should comprise at least a therapeutically effective amount of the 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.
In this context, 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 (AUCIN×doseIN)/(AUCIV×doseIV)×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). The relative bioavailability of Polypeptides of the Invention is determined by comparing the concentration vs. time plot of Polypeptides of the Invention after intramuscular (IM) or subcutaneous (SC) administration with the concentration vs. time plot of Polypeptides of the Invention after oral (IN) administration. The relative bioavailability of Polypeptides of the Invention is defined as (AUCIN×doseIN)/(AUCSC/IM×doseSC/IM)×100. Accordingly, in order to be equipotent to the injection route, oral administration will appropriately be effected so as to give a dosage rate of the order of 1 to 100 times, preferably 1 to 50 times, more preferably 1 to 20 times, even more preferably 1 to 10 times the dosage required for treatment via injection, also depending on the frequency of the oral application.
The amount of active compound will generally be chosen to provide effective treatment on administration once a day or once a week or once a month. Alternatively, dosages may be split over a series of e.g. 1 to 4 applications taken at intervals during the day, week or month. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple takings of a plurality of pills or capsules. To maintain more consistent or normalized therapeutic levels of the Polypeptide of the Invention, it may be advisable that the Composition of the Invention is repeatedly administered to the subject, for example one, two or more times within a 24 hour period, four or more times within a 24 hour period, six or more times within a 24 hour period, or eight or more times within a 24 hour period. An administration regimen could include long-term, daily, weekly or monthly treatment. By “long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. The clinician will generally be able to determine a suitable daily, weekly or monthly dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.
The final determination of the effective dosage will be based on animal model studies, followed up by human clinical trials, and is guided by determining effective dosages and oral administration protocols that significantly reduce the occurrence or severity of the targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Ultimately, the dosage of 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.
As a non-limiting example, 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.
The proportion of each further component in the oral composition of the invention may vary depending on the components used. For example, but without being limiting, the amount of enteric coating may be in the range of from 0.1 to 99.9%, preferably 1 to 20% by weight of the total weight of the composition. When present, the amount of permeability enhancer may be in the range from about 0.01 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.
In addition to the concentration of the different compounds in the composition of the invention, the total delivery weight is important to consider as well. The delivery weight is relatively high for oral compositions and may be up to 1 g or more. Suitable delivery weights will be clear to a person skilled in the art of pharmacology.
The present invention further provides a method for the preparation of a composition mixing the Polypeptides of the Invention, e.g. the single variable domains, Nanobodies, the domain antibodies, the single domain antibodies or the dAbs and the pharmaceutically acceptable excipients (as proposed herein, e.g. protease inhibitors, slow release matrices, and/or permeability enhancer) and thus resulting in a powder that is then further e.g. filled into capsules, preferably enterically coated capsules. Alternatively, said powder comprising the 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. E.g. the Polypeptides of the Invention, e.g. Nanobodies, may be admixed with the protease inhibitors, slow release matrices, and/or permeability enhancer, optionally with further ingredients, additives and/or agents as indicated above. The Polypeptides of the Invention, e.g. Nanobodies, may be in solution e.g. an aqueous or alcoholic solution when being mixed with the protease inhibitors, slow release matrices, and/or permeability enhancer and the solvent evaporated, e.g. under freeze-drying or spray drying. Such drying may be effected under the conventional conditions. Alternatively the dry mixtures may be compacted and/or granulated and then be pulverized and/or sieved. If desired the compacted composition may be further coated. According to a preferred 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.
Alternatively, 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.
For administration of a liquid, for example, such compositions will suitably be put up in a container provided with a conventional dropper/closure device, e.g. comprising a pipette or the like, preferably delivering a substantially fixed volume of composition/drop.
If desired a powder or liquid may be filled into a soft or hard capsule adapted for oral administration. The powder may be sieved before filled into the capsules such as gelatine capsules, preferably an enterically coated capsule.
The Pharmaceutical Composition of the Invention is formulated for oral administration and for delivery of the 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. This means that there is a sufficient amount of functional (i.e. active or not inactivated) Polypeptide of the Invention, e.g. the Nanobodies, present in the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) to provide the desired therapeutic effect (i.e. to elicit the desired activity or the desired biological, prophylactic or therapeutic response in the subject receiving said Polypeptide of the Invention, e.g. the Nanobodies. The bioavailability of the Polypeptides of the Invention, e.g. the Nanobodies, in the blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) and/or in the brain following administration of the composition of the invention is determined by measuring the pharmacokinetic parameters Cmax (peak concentration), AUC (area under concentration vs. time curve) and/or Tmax (time to maximal blood concentration), which are well known to those skilled in the art (Laursen et al., Eur. J. Endocrinology, 1996; 135: 309-315). The bioavailability of the Polypeptide of the Invention, e.g. the Nanobodies, may be determined in any conventional mariner, e.g. by radioimmunoassay.
“Cmax”, as used in the present invention, 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. In an embodiment 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. 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, 100 ng or more of Polypeptide of the Invention, e.g. the Nanobodies, per ml of blood.
In another embodiment, 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. 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, 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.
In another embodiment of the invention, following oral administration of Polypeptide of the Invention, said polypeptide reaches a Cmax in blood of at least 1% of the Cmax that is reached following parenteral administration of the same amount of the Polypeptide of the Invention. In a further 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”, as used in the present invention, 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. In an embodiment of the invention, the Polypeptide of the Invention reaches the bloodstream with a Tmax of less than 120 minutes. In a further embodiment, 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.
In an embodiment, 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. In another embodiment, 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. 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 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of the composition of the invention.
In another embodiment, 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. 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 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. In another embodiment, 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. 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 90, 60, 50, 40, 30, 20, 10, or 5 minutes following oral administration of a dose of 5 mg/kg body weight of said Polypeptide of the Invention.
The “area under the curve (AUC)”, as used in the present invention, is the area under the curve in a plot of concentration of the 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. Mathematically, this value is a measure of the integral of the instantaneous concentrations during a time interval. AUG is usually given for the time interval zero to infinity, and other time intervals are indicated (for example AUC (t1,t2) where t1 and t2 are the starting and finishing times for the interval). Clearly blood (and/or another selected physiological compartment, tissue and/or organ such as e.g. the kidney, bladder and/or lung) 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. In an embodiment 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. In a further embodiment, 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 μg/ml/minute of the Polypeptide of the Invention.
In another embodiment 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. In a further embodiment, 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 μg/ml/minute Polypeptide of the Invention per ml of blood.
As discussed above, in an embodiment 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 1% compared to parenteral administration of said Polypeptide of the Invention. In a further embodiment, 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. Preferably 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. In a preferred embodiment of the invention, the Polypeptide of the Invention provides the prevention and/or treatment of a selected disease or condition in said subject. Accordingly, another aspect of the invention relates to a method for the prevention and/or treatment of a subject in need of a 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.
In the context of the present invention, the term “prevention and/or treatment” not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.
The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and/or disorder.
The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a 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.
In another embodiment, 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-α, IgE, IFN-γ, MMP-12, EGFR, CEA, H. pylori, M. tuberculosis, influenza, β-amyloid, vWF, IL-6, IL-6R, PDK1, CD40, OVA, VSG, S. typhimurium, Rotavirus, Brucella, parathyroid hormone-derived peptides.
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. In an embodiment, 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. Accordingly, 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.
In another embodiment, 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. Accordingly, 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.
In another preferred embodiment, 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. Accordingly, 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.
In another embodiment, 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, 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. The invention also relates to a method for the prevention and/or treatment of a disease or disorder such as an autoimmune disease (such as e.g. rheumatoid arthritis or Inflammatory Bowel Disease), said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of the Invention that is directed against TNF and/or of a composition comprising the same. Accordingly, 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).
In another embodiment, 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. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction), said method comprising orally administering to said subject a therapeutically effective amount of a Polypeptide of the Invention that is directed against vWF and/or of a composition comprising the same. Accordingly, the present invention also relates to the composition of the invention, wherein the 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. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction).
In another embodiment, 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, 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 a disease or disorder associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex (such as e.g. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, 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 Invention that is directed against IL-6, IL-6R and/or IL-6/IL-6R complex and/or of a composition comprising the same. Accordingly, 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. sepsis, various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases and disorder such, as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus).
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.
Generally, the treatment regimen will comprise the oral administration of one or more Nanobodies, polypeptides or proteins of the invention, or of one or more compositions comprising the same, in one or more therapeutically effective amounts or doses. The specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.
The Nanobodies and 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. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.
When a second active substances or principles is to be used as part of a combined treatment regimen, it can be administered via the same oral route of administration or via a different route of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are administered to be simultaneously via the same oral route of administration, they may be administered as different formulations or compositions or part of a combined formulation or composition, as will be clear to the skilled person.
Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.
The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and or a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.
Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.
The invention also relates to the use of a 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). 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. the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischaemia, cardioembolism, peripheral vascular disease, restenosis and myocardial infarction). The invention also relates to the use of a 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 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), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer, bone resorption (osteoporosis), cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus).
As discussed above, oral administration of one or more 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. In addition to the prophylactic and therapeutic response as discussed above, the Nanobodies, polypeptides and proteins of the invention may also induce other activities and biological responses. In a preferred embodiment, 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 Invention and/or a composition comprising the same. In an embodiment of the invention, a diagnostic method is provided 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. 1996; 109: 93-96), Sandrock et al. (Nucl. Med. Commun. 1996; 17: 311-316), and Mariani et al. (Cancer 1997; 15: 2484-2489). These in vivo imaging methods may allow the localization and possibly quantification a certain target, for example, by use of a labeled Polypeptide of the Invention, specifically recognizing said target. In vivo multiphoton microscopy (Bacskai et al., J. Cereb. Blood Flow Metab. 2001; 22: 1035-1041) can be used to image the presence of a certain target with labeled Polypeptides of the Invention specific for the target.
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. Such detectable labels have been well developed in the field of immunoassays and, in general, almost any label useful in such methods can be applied to the method of the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means. Useful labels in the present invention include but are not limited to magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g. fluorescein isothiocyanate, Texas red, rhodamine, Cy3, Cy5, Cy5.5, Alexi 647 and derivatives), radiolabels (e.g. 3H, 125I, 35S, 14C, 32P or 99mTc), enzymes (e.g. horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
The label may be coupled directly or indirectly to the 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.
Finally, although the use of the Polypeptides of the Invention (as defined herein, e.g. the Nanobodies and/or constructs comprising said Nanobodies) is much preferred, it will be clear that on the basis of the description herein, the skilled person will also understand that other (single) domain antibodies, as well as polypeptides and proteins comprising such (single) domain antibodies (in which the terms “domain antibody”, “single domain antibody” and “dAb” have their usual meaning in the art) are also encompassed within the scope of the present invention.
The invention will now be further described by means of the following non-limiting experimental part.
The erythropoietin receptor (EpoR) and the growth hormone receptor (GHR) belong to the cytokine receptor type I superfamily for which signaling is known to be triggered by ligand-induced receptor homodimerization and mediated by cytoplasmic protein tyrosin kinases of the Jak family (Watowich, 1999; Frank, 2002; Brooks et al, 2007). In the case of the EpoR, upon binding of erythropoietin (Epo), receptor dimerization and activation of the signal transduction pathway lead to erythtroid cell survival, proliferation and differentiation. The GHR dimerization and signaling induced by the growth hormone is the key regulator of postnatal growth and has important actions on metabolism, reproductive, gastrointestinal, cardiovascular, hepto-biliary and renal systems (Brooks et 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. 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). For the related GH receptor, a variety of agonist monoclonal antibodies have been also reported (Rowlinson et al., 1998).
Structure of EpoR and GHR and Interaction with Natural Ligands
The EpoR and GHR, as other members of the cytokine receptor type I superfamily, are cell surface proteins composed of an NH2-terminal ligand binding domain, a COOH-terminal cytoplasmic region and a single membrane-spanning domain. Conserved features of the extracellular domain include two pairs of cysteine residues and a ‘WSXWS” motif with characteristic spacing.
While receptor dimerization is a common activation mechanism for this family of cytokine receptors there seem to be small differences between the protein folding pathways and/or three-dimensional structures of individual receptors which dictate their potential to be covalently dimerized by disulfide bridges.
The classical model for activation of GHR is described as the formation of a ligand-receptor complex made up of one GH molecule and two GHR (GH:2 GHR). 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). Other studies revealed that the receptor can be found as a dimer on the surface of the cell in the absence of GH leading to a paradigm shift whereby most evidences support a model of GH binding to a constitutively homodimerised GHR which causes the recognition of the intracellular domains resulting in the activation of the signal transduction (Brooks et al., 2007; Waters et al, 2006). This current model of signaling also applies to the closely related. EpoR (Watowich, 1999). Early studies of the EpoR/Epo complex suggested a 1:1 stoichiometry, although later studies demonstrated a 2:1 stoichiometry showing two binding sites of Epo for the extracellular domain of the receptor. Interaction of the first site is of high affinity (dissociation constant 1 nM) while the second binding interaction is much weaker (1 uM). There are also some reports that evidence for preformed dimers of EpoR before ligand activation (Livnah et al., 1999; Lu et al., 2006). In that case the binding of Epo changes the orientation of the two receptor subunits, transmitting a conformational change through the transmembrane domains leading to activation of JAK2 kinase and induction of proliferation and survival signals.
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).
Two llamas (215 and 216) will be immunized, according to standard protocols, with 6 boosts of a cocktail 152 containing:
Recombinant mouse EpoR/Fc Chimera (R&D Systems Cat No 1390-ER).
Blood will be collected from these animals 4 and 8 days after boost 6.
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. Following incubation with the phage libraries and extensive washing, bound phage will be totally eluted with trypsin and specifically eluted with Epo. If necessary the eluted phage are amplified and applied in a second round of selection on 5 ug/ml, 0.5 ug/ml, 0.05 ug/ml and 0 ug/ml (control) immobilized EpoR/Fc.
Optionally, 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. 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).
Individual colonies of E. coli TG1 infected with the obtained eluted phage pools will be 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).
In order to determine binding specificity to EpoR, 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. After incubation and a wash step, 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 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.
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. For this, 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 (Centaur 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-containing periplasmic extracts (P.E.) instead of phage and detecting with a mouse anti-myc antibody and an anti mouse-HRP antibody.
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. For this, 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. In fact, these non-neutralizing Nanobodies will be used preferably for the construction of agonistic construct comprising e.g. 2 EpoR non-neutralizing Nanobodies. Thus, e.g. various constructs will be generated comprising 2 EpoR non-neutralizing Nanobodies that are identified above, e.g. linked by a 9 Gly linker (see e.g. WO 2007/104529).
Reference is made to the assay described in J. Tavernier et al, supra. In short, a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric “bait” construct, e.g. a construct wherein the extracellular domain of the leptin receptor (LR) is replaced by the murine (see vector pCEL 1f in Tavernier's publication above) or human (see vector pSEL1 same publication) ligand binding extracellular Epo-R gene; b) infection of cells stably expressing the “bait” with above identified bivalent Nanobody constructs; and e) stimulation and selection using puromycin with results in surviving clones that express the agonistically acting bivalent Nanobody construct (confirmation that bivalent Nanobody construct is indeed acting agonistically).
Two llamas (215 and 216) will be immunized, according to standard protocols, with 6 boosts of a cocktail 152 containing:
Recombinant mouse GHR/Fc Chimera (R&D Systems Cat No 1360-GR).
Blood will be collected from these animals 4 and 8 days after boost 6.
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 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 GHR/Fc the phage will be pre-incubated with 250 ug/ml human IgG. Following incubation with the phage libraries and extensive washing, bound phage will be totally eluted with trypsin and specifically eluted with GH. If necessary the eluted phage were amplified and applied in a second round of selection on 5 ug/ml, 0.5 ug/ml, 0.05 ug/ml and 0 ug/ml (control) immobilized GHR/Fc. Individual colonies of E. coli TG1 infected with the obtained eluted phage pools will be grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).
Optionally, the Phage libraries 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. 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).
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. 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 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 GH and/or no phage. The same kind of competition assay could be performed using Nanobody-containing periplasmic extracts (P.E.) instead of phage and detecting with a mouse anti-myc antibody and a anti mouse-HRP antibody.
Clones tested positive in the GHR binding assay (including clones selected for protease resistancy) will be screened for their ability not to block GH binding to GHR/Fc. For this, 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. In fact, these non-neutralizing Nanobodies will be used preferably for the construction of agonistic construct comprising e.g. 2 GHR non-neutralizing Nanobodies. Thus, e.g. various constructs will be generated comprising 2 GHR non-neutralizing Nanobodies that are identified above, e.g. linked by a 9 Gly linker (see e.g. WO 2007/104529).
Reference is made to the assay described in J. Tavernier et al, supra. In short, a typical screening or test assay comprises the following three successive steps: a) stable transfection of the chimeric “bait” construct, e.g. a construct wherein the extracellular domain of the leptin receptor (LR) is replaced by the murine (see vector pCEL if in Tavernier's publication above) or human (see vector pSEL1 same publication) ligand binding extracellular Epo-R gene; b) infection of cells stably expressing the “bait” with above identified bivalent Nanobody constructs; and c) stimulation and selection using puromycin with results in surviving clones that express the agonistically acting bivalent Nanobody construct (confirmation that bivalent Nanobody construct is indeed acting agonistically).
Receptor dimerization in GH and Erythropoietin action—It takes two to tango, but how? Endocrinology (2002). 143: 2-10.
Activation of erythropoietin signaling by receptor dimerization. The International Journal of Biochemistry & Cell biology (1999). 31: 1075-1088.
Growth hormone receptor; mechanism of action. The International Journal of Biochemistry & Cell biology (2007). Doi: 10.1016/j. biocel. Jul. 8, 2007.
Negative regulation of growth hormone receptor signaling. Molecular Endocrinology (2006). 20: 241-253.
New insights into growth hormone action. Journal of Molecular Endocrinology (2006). 36: 1-7.
The major histocompatibility complex class I-related receptor FcRn was first identified as the receptor that transports maternal IgGs from mother to young via the neonatal intestine. However recent data have indicated that the neonatal receptor is also responsible for rescuing IgG and albumin from degradation and therefore prolong their half-lives (Andersen et 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).
The role of FcRn as an IgG transporter opens the opportunity to generate new therapeutics for modulation of IgG levels, as it is desired in the case of autoimmune diseases. Because the transport and protection of IgG are dependent on its Fc-domain, it can be proposed that small molecules or peptides with therapeutic activities could be fused to Fc fragments and therefore delivered across the epithelium and have long circulating long lives. Moreover, the fact that FcRn is expressed on many epithelial surfaces in adult humans including the lungs (Spiekermann et al., 2002; Bitonti and Dumont, 2006), suggests that FcRn transport pathway could be used as a delivery system of therapeutic agents by non-invasive means (i.e. aerosols administered into the lungs using normal breathing maneuvers).
FcRn comprises a heterodimer of beta2-microglobulin and a 45 to 53 KDa protein. All three extracellular and membrane domains of FcRn share homology with the corresponding regions of major histocompatibility complex (MHC) class I molecules, with much less homology between the cytoplasmic domains. The X-ray crystallographic structure of the extracellular domains of FcRn confirmed that it is structurally similar to MHC class I molecules (Ghetie and Ward, 2000).
The FcRn-IgG interaction depends on conserved histidine residues in the IgG-Fc part that interact with negatively charged residues in the beta-2 domain of the hFcRn heavy chain. Recent studies showed that conserved H166 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).
The fact that FcRn regulates IgG homeostasis, modulation of FcRn function and/or expression might be an effective approach for the treatment of autoimmune diseases. It has been suggested that deregulation of FcRn expression may be involved in situations in which hypercatabolism is observed, such as after burns and in myotonic 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).
Identification of Nanobodies Binding hFcRn:
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 mutmix (intact soluble FcRn, heavy chain and beta2-microglobulin).
For animal 153, blood was collected 4 and 7 days after boost 6. In addition, approximately 1 g of lymph node was collected from this animal 4 days after boost 6
For animal 154, blood was collected 22 days after boost 3.
Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
Phage library 153 was used for selection at pH 5 on hFcRn heavy chain (hFcRn HC) while phage library 154 was used for selection at pH 5 on shFcRn (heavy chain and beta2-microglobulin). Both hFcRn proteins were immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control) in PBS at pH 7.4. After 2 hours blocking with 4% Marvel PBS the plates were washed several times with PCA buffer/Tween pH 5.1 (10 mM Sodium citrate+10 mM Sodium phosphate+10 mM Sodium acetate+115 mM NaCl/Tween pH 5.1) To minimize the number of phage binding to the albumin binding site of the FcRn protein the phage was pre-incubated with 250 ug/ml human serum albumin in 2% Marvel PCA buffer pH 5.1. Following incubation with the phage libraries and extensive washing with pH 5.1 buffer, bound phage was eluted with trypsin. The eluted phage were amplified and applied in a second round of pH 5 selection on 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control) immobilized hFcRn proteins. To minimize the number of phage binding to the albumin binding site of the FcRn protein the phage was pre-incubated with 250 ug/ml human serum albumin in 2% Marvel PCA buffer pH 5.1. Individual colonies obtained from the eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).
In order to determine binding specificity to hFcRn, the clones were tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to hFcRn HC was tested. Shortly, 0.5 ug/ml hFcRn HC was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. After washing with PCA ph 5.1 buffer, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PCA pH 5.1 were allowed to bind to the immobilized antigen. After incubation and several wash steps with pH 5.1 buffer, phage binding was revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat# 279421.01) in 1% Marvel PCA pH 5.1.
The same ELISA assay was performed at neutral pH by using PCA pH 7.4 buffer. Binding specificity was determined based on OD values compared to controls wells having received an irrelevant phage or no phage.
Perspective-FcRn transport albumin: relevance to immunology and medicine. TRENDS in Immunology (2006). 27: 343-348
Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu. Rev. Immunol. (2000) 18: 739-766
A passionate kiss, then run: exocytosis and recycling of IgG by FcRn. TRENDS in cell biology (2005). 15: 5-9.
Two llamas (097 and 098) were immunized with 6 boosts of R&D Systems Cat #2717-PG, which is the ectodomain of human pIgR, according to standard protocols. Blood was collected from these animals after 7 days after boost 6 and 10 days after boost 6.
Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use.
Phage libraries from llama's 097 and 098 were used for selections for two rounds on ectodomain of pIgR (R&D Systems Cat #2717-PG). pIgR was immobilized directly on Nunc Maxisorp ELISA plates at 5 microg/ml or 1 ug/ml and 0 ug/ml (low control) for the first round of selection and 5 microg/ml or 0.5 ug/ml and 0 ug/ml (low control) for the second round of selection. Binding phages were retrieved from both first and second selection rounds using trypsin elution, IgA specific elution and BSA specific elution (neg. control).
Specific elution was performed by incubating the wells with 150 ug/ml IgA for 1 hour, thereby replacing Nanobodies binding on the IgA binding spot of pIgR.
For the second round of selection phages from the output of the first round of selection eluted with IgA were used.
Output of both R1 and R2 selections were analyzed for enrichment factor (# phage present in eluate relative to control). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 ul) were prepared according to standard methods (see for example the prior art published and applications filed by applicant).
In order to determine binding specificity to pIgR, the clones were tested in an ELISA binding assay setup.
In short, 5 ug/ml pIgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 ul of periplasmic extract containing Nanobody of the different clones in 100 ul 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). Overall more than 70% of the selected clones were able to bind to pIgR with some specificity (signal more than 2× above background).
In order to determine IgA competition efficiency of pIgR binding Nanobodies the positive clones of the binding assay were tested in an ELISA competition assay setup. In short, 5 ug/ml pIgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 1 ug/ml of IgA was preincubated with 10 ul of periplasmic extract containing Nanobody of the different clones and a control with only IgA (high control). The IgA was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, IgA binding was revealed using a rabbit-anti-IgA secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control).
In order to determine IgA competition efficiency of IgA competitive Nanobodies clones of the binding assay were tested in an ELISA competition assay setup.
In short, 5 ug/ml pIgR ectodomain was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 1 ug/ml of IgA was preincubated with a dilution series of purified Nanobody and a control with only IgA (high control). The IgA was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, IgA binding was revealed using a rabbit-anti-IgA secondary antibody (Serotec cat #AHP525H), which was after a wash step detected with a HRP-conjugated donkey-anti-rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control) and two Nanobodies that can bind to pIgR but do not compete for IgA binding.
The results confirm that clones 1D2, 1D7, 1E7, 4B11 and 4D9 have a antagonistic effect on IgA binding to pIgR. 1D2 and 4B11 are inferior in this to the other clones.
In order to determine binding specificity to pIgR in cells Nanobody 4B11 was tested in an immunofluorescence setup (adapted from Klapisz 2002).
In short, MDCK cells overexpressing human pIgR were grown on glass cover slips and free binding sites were blocked using precooled 4% Marvel in PBS at 4C. Next, 3 uM of purified Nanobody in 50 ul 2% Marvel PBS was allowed to bind to the cells at 4C. After incubation and a wash step, cells were fixed using 4% paraformaldehyde. Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a Cy2-conjugated donkey-anti-mouse antibody. For reference nuclei were stained using DAPI. Fluorescence signal was detected under an epifluorescence microscope (Leica) attached to a cooled CCD camera (Micromax, Princeton Instruments). The pictures were taken using Metamorph and the final figures were obtained using the NIH Image and Adobe Photoshop programs. Pictures show that Nanobody 4B11, 1D2 and 1E7 can bind to human pIgR in a cellular environment.
In order to check whether Nanobodies are able to bind the hpIgR in its native form, an immunoprecipitation experiment was performed nanobodies, containing a His-tag, were allowed to bind to hpIgR in cell lysates and fished out with talon beads. The hpIgR was detected on blot with a-hSC and DAG-PO. As a control the VHHs bound to the beads were detected with a-myc and DAM-PO. The result of this immunoprecipitation experiment clearly shows that the VHHs: 1D2, 4B11, 4B7 and 1D7 are able to bind to hpIgR in cell lysates. The receptor could be detected in the four lanes containing lysates with the hpIgR binding VHHs. Empty talon beads and nanobodies directed against the EGF receptor were not able to detect the receptor and the receptor was also not detected in lysates of untransfected MDCK cells. The lysate control shows that the nanobodies are able to enrich for this receptor out of cell lysate. The binding of the Nanobodies to the talon beads was checked and this shows that indeed all lanes contained beads with bound VHHs, except for the empty beads and the VHHs were also not present in the cell lysate.
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 pIgR from the basolateral to the apical side.
The transcytosis assay is performed with fully polarized MDCK cells, seeded on 1 cm2, 0.4 μm 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.
106 phages were added to the basolateral chamber of the Transwell-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.
This showed that phages 1D2 and 4B7 are transcytosed over MDCK cells by hpIgR in a transwell assay from basolateral to apical.
After approval of the Ethical Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), 2 llamas (117, 118) were alternately immunized with 6 intramuscular injections at weekly interval with human serum albumin and a mixture of mouse serum albumin, cynomolgus serum albumin and baboon serum albumin, according to standard protocols.
When an appropriate immune response was induced in llama, four days after the last antigen injection, a 150 ml blood sample is collected and peripheral blood lymphocytes (PBLs) were purified by a density gradient centrifugation on Ficoll-Paque™ according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage is prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored at 4° C. for further use.
In a first selection, human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates were blocked with 4% Marvel in PBS for 2 h at RT. After 3 washes with PBST, phages were added in 4% Marvel/PBS and incubated for 1 h at RT. Following extensive washing, bound phage was eluted with 0.1 M triethanolamine (TEA) and neutralized with 1M Tris-HCl pH 7.5.
To enrich for conditional binders, said binders with a pH sensitive interaction, phage libraries were incubated with antigen at physiological pH and eluted at acidic pH as follows.
In a first selection, human serum albumin (Sigma A-8763) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates are blocked with 4% Marvel in PBS pH 7.3 for 2 h 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 2 h at RT. Unbound phages were removed by 10 washes with PBST pH7.3, followed by 2 washes with PBS pH5.8. Bound phage was eluted with PBS pH5.8 for 30 min at RT and neutralized with 1M Tris-HCl pH. 7.5.
In a second selection, phage libraries were incubated for 2 h at RT with human serum albumin in 2% Marvell/CPA buffer (10 mM sodium citrate+10 mM sodium phosphate+10 mM sodium acetate+115 mM NaCl) adjusted to pH 7.3. Unbound phages were removed by 10 washes with CPA/0.05% Tween20 (CPAT) pH7.3, followed by 2 washes with CPAT pH5.8. Bound phage was eluted with CPA pH5.8 for 30 min at RT and neutralized with 1M Tris-HCl pH 7.
In a third selection strategy, phage libraries were incubated for 2 h at RT with human serum albumin in 2% Marvell/CPA pH 5.8. Unbound phages are removed by 10 washes with CPAT pH 5.8, followed by 2 washes with CPA pH 7.3. Bound phage was eluted with 1 mg/ml trypsin/CPA pH 7.3 for 30 min at RT.
In a fourth selection strategy, phage libraries were incubated for 2 h 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 1 mg/ml trypsin/CPA pH 7.3 for 30 min at RT.
In all selections, enrichment was observed. The output from each selection was re-cloned as a pool into the expression vector pAX51. Colonies were picked and grown in 96 deep-well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant).
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 MCA1396B) detected with Streptavidin-HRP (DakoCytomation #P0397). The signal was developed by adding TMB substrate solution (Pierce 34021) and detected at a wavelength of 450 nm. A high hit rate of positive clones can already be obtained after panning round 1.
To enrich for conditional binders, said binders with a pH sensitive interaction, phage libraries may be incubated with antigen at physiological pH and eluted at acidic pH as follows.
In a first selection strategy, human serum albumin (Sigma A-8763) is coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 100 μg/ml overnight (ON) at room temperature (RT). Plates are blocked with 4% Marvel in PBS pH 7.3 for 2 h 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 2 h at RT. Unbound phages were removed by 10 washes with PBST pH7.3, followed by 2 washes with PBS pH5.8. Bound phage was eluted with PBS pH5.8 for 30 min at RT and neutralized with 1M Tris-HCl pH 7.5.
In a second selection strategy, phage libraries were incubated for 2 h at RT with human serum albumin in 2% Marvell/CPA buffer (10 mM sodium citrate+10 mM sodium phosphate+10 mM sodium acetate+115 mM NaCl) adjusted to pH 7.3. Unbound phages were removed by 10 washes with CPA/0.05% Tween20 (CPAT) pH7.3, followed by 2 washes with CPAT pH5.8. Bound phage was eluted with CPA pH5.8 for 30 min at RT and neutralized with 1M Tris-HCl pH 7.
In a third selection strategy, phage libraries were incubated for 2 h at RT with human serum albumin in 2% Marvell/CPA pH5.8. Unbound phages are removed by 10 washes with CPAT pH5.8, followed by 2 washes with CPA pH 7.3. Bound phage is eluted with 1 mg/ml trypsin/CPA pH 7.3 for 30 min at RT.
In a fourth selection strategy, phage libraries were incubated for 2 h 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 1 mg/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 (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) are prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).
Human serum albumin was immobilized on a CM5 sensor chip surface via amine coupling using NHS/EDC for activation and ethanolamine for deactivation (Biacore amine coupling kit)
Approximately 1000RU of human serum albumin was immobilized. Experiments were performed at 25° C. The buffers used for the pH dependent binding of Nanobodies to albumin (Biacore) are as follows: 10 mM Sodium citrate (Na3C6H5O7)+10 mM Sodium phosphate (Na2HPO4)+10 mM Sodium Acetate (CH3COONa)+115 mM 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 1 min at a flow rate of 45 ul/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 T100 evaluation software.
The off rate of different Nanobodies at pH7 and pH5 is documented in Table E-1. The majority of the Nanobodies (4A2, 4A6, 4B5, 4B6, 4B8, 4C3, 4C4, 4C5, 4C8, 4C9, 4D3, 4D4, 4D7 ad 4D10 have a faster off rate at pH 5 compared with pH 7 (2-6 fold difference in off rate). The Nanobody 4A9 has a slower off-rate at pH 5 compared to pH 7 (0.54 fold difference in off rate). For other Nanobodies including 4C12, 4B1, 4B10, IL6R202, Alb-8, and 4D5, binding to antigen does not change at different pH.
Direct screening of nanobody repertoires for conditional binding to antigen can thus be used.
To screen Nanobodies for their conditional binding to albumin, a binding ELISA can also be performed with two representative conditions, pH 5.8 and pH7.3 and the relative binding strength determined. Maxisorb micro titer plates (Nunc, Article No. 430341) were coated overnight at 4° C. with 100 μl of a 1 μg/ml solution human serum albumin in bicarbonate buffer (50 mM, pH 9.6). After coating, the plates were washed three times with PBS containing 0.05% Tween20 (PBST) and blocked for 2 hours at room temperature (RT) with PBS containing 2% Marvel (PBSM). After the blocking step, the coated plates were washed 2 times with PBST pH 5.8, and a ten-fold dilution aliquot of each periplasmic sample in PBSM pH5.8 (100 μl) is transferred to the coated plates and allowed to bind for 1 hour at RT. After sample incubation, the plates were washed five times with PBST and incubated for 1 hour at RT with 100 μl of a 1:1000 dilution of mouse anti-myc antibody in 2% PBSM. After 1 hour at RT, the plates were washed five times with PBST and incubated with 100 μl of a 1:1000 dilution of a goat anti-mouse antibody conjugated with horseradish peroxidase. After 1 hour, plates were washed five times with PBST and incubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20 minutes, the reaction was stopped with 100 μl H2SO4. The absorbance of each well was measured at 450 nm.
92 periplasmic extracts for each of the conditional selection strategies described herein, are analyzed in this ELISA. 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.
Upon 1 round of selection on human serum albumin, followed by total elution, Nanobodies are identified that either conditionally bind to albumin at acidic pH (n=16) or at neutral pH (n=19). Driving the selection conditions towards conditional binding, results in a higher ratio of conditionally binding nanobodies (n=23).
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).
The conditional pH-binding properties of the anti-FcRn or pIgR Nanobody or dAbs within the multispecific nanobody formats (e.g. are evaluated via surface plasmon resonance (BIAcore), e.g. a conditional binder as disclosed in this application is linked to one or more nanobody or dAbs binding to one or more protein target(s), e.g. is linked to 2 Nanobodies directed against Epo-R or HGR. Cross-reactivity to cynomolgus serum albumin is also assessed. Human and cynomolgus FcRn or pIgR are immobilized on a CM5 sensor chip surface via amine coupling using NHS/EDC for activation and ethanolamine for deactivation (Biacore Amine Coupling Kit)
Experiments are performed at 25° C. The buffers used for the pH dependent binding of Nanobodies to FcRn or pIgR (Biacore) are as follows: 10 mM Sodium citrate (Na3C6H5O7)+10 mM Sodium phosphate (Na2HPO4)+10 mM Sodium Acetate (CH3COONa)+115 mM NaCl. This mixture is brought to pH7, pH6 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 1 min at a flow rate of 45 ul/min over the activated and reference surfaces. Those surfaces are regenerated with a 3s pulse of glycine-HCl pH1.5+0.1% P20. Evaluation is done using Biacore T100 evaluation software.
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 pIgR 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 pIgR binding block, again linked e.g. via a 9 amino acid Gly/Ser linker) is administered intravenously by bolus injection (1.0 ml/kg, approximately 30 sec) in the vena cephalica of the left or right arm to obtain a dose of 2.0 mg/kg. The Nanobody concentration in the plasma samples is determined via ELISA.
The concentration in the plasma samples is determined as follows:
Maxisorb micro titer plates (Nunc, Article No. 430341) are coated overnight at 4° C. with 100 μl of a 5 μg/ml solution of the Polypeptide of the Invention in bicarbonate buffer (50 mM, pH 9.6). After coating, the plates are washed three times with PBS containing 0.1% Tween20 and blocked for 2 hours at room temperature (RT) with PBS containing 1% casein (250 μl/well). Plasma samples and serial dilutions of polypeptide-standards (spiked in 100% pooled blank cynomolgus plasma) are diluted in PBS in a separate non-coated plate (Nunc, Article No. 249944) to obtain the desired concentration/dilution in a final sample matrix consisting of 10% pooled cynomolgus plasma in PBS. All pre-dilutions are incubated for 30 minutes at RT in the non-coated plate. After the blocking step, the coated plates are washed three times (PBS containing 0.1% Tween20), and an aliquot of each sample dilution (100 μl) is transferred to the coated plates and allowed to bind for 1 hour at RT. After sample incubation, the plates are washed three times (PBS containing 0.1% Tween20) and incubated for 1 hour at RT with 100 μl of a 100 ng/ml solution of sIL6R in PBS (Peprotech, Article No. 20006R). After 1 hour at RT, the plates are washed three times (PBS containing 0.1% Tween20) and incubated with 100 μl of a 250 ng/ml solution of a biotinylated polyclonal anti-Polypeptide of the Invention in PBS containing 1% casein (R&D systems, Article No. BAF227). After incubation for 30 minutes (RT), plates are washed three times (PBS containing 0.1% Tween20) and incubated for 30 minutes (RT) with 100 μl of a 1/5000 dilution (in PBS containing 1% casein) of streptavidine conjugated with horseradish peroxidase (DaktoCytomation, Article No. P0397). After 30 minutes, plates are washed three times (PBS containing 0.1% Tween20) and incubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20 minutes, the reaction is stopped with 100 μl HCl (1N). The absorbance of each well is measured at 450 nm (Tecan Sunrise spectrophotometer), and corrected for absorbance at 620 nm. This assay measures free Polypeptide of the Invention as well as Polypeptide of the Invention bound Polypeptide of the Invention. Concentration in each plasma sample is determined based on a sigmoid standard curve with variable slope of the respective Polypeptide of the Invention.
Each individual plasma sample is analyzed in two independent assays and an average plasma concentration is calculated for pharmacokinetic data analysis.
All parameters are calculated with two-compartmental modeling, with elimination from the central compartment.
For the purposes of illustrating this invention in e.g. a monkey or mouse, the enteric coating material is selected from HPMC-AS (pH 5.5), CAT (pH 5.5) and Eudragit L (pH 5.5), most preferably Eudragit L (pH 5.5).
For use in the human, the enteric coating material preferably may be one which will provide for release of polypeptide at about pH 6.0-6.5 such as, for example, CAP and HPMC-AS.
The enterocoating is carried out by methods known per se in the art, e.g., according to Remington Pharmaceutical Sciences, p. 1614-1615 (1975, 15th Ed., Mack Pub. Co.) and Theory and Practice of Industrial Pharmacy, Lackman, Liberman & Caning, p. 116-117, 371-374 (1976, 2nd Ed.). The enteric micro-encapsulation process is also known (Theory and Practice of Industrial Pharmacy ibid, pp. 420-438). See also Remington Pharmaceutical Sciences, p. 1637 (1985, 17th Ed., Mack Pub. Co.). Typically, the amount of enteric coating material used preferably is in the range about 10-20 mg per 500 cm.sup.2 of surface area of capsule or tablet, especially of capsule as produced in the actual examples described herein below. The amount of enteric coating material broadly is in the range of about 1-5000 mg/capsule, more preferably about 10-1000 mg/capsule, most preferably about 50-100 mg/capsule.
A solution comprising the 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 pIgR binding Nanobodies (preferably pH dependent binding, e.g. binding at pH 6 or less but not or to a much lower extend at pH 7 and more)) is filled up into enteric coated capsules and used within a short time, e.g. within a week or day for the in vivo experiment as e.g. presented in the below examples.
A liquid formulation will generally consist of a solution or suspension containing the biologically active polypeptide, e.g. the 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-miscible solvents such as, for example, ethanol, glycerin, propylene gylcol and sorbitol, or mixtures of any of the foregoing.
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 Gly linker), optionally comprising a pH independent or pH dependent anti-mouse FcRn or pIgR Nanobody (i.e. binding at gut pH, about pH 6, but released at blood pH7 or more) are injected intraperitoneally, gauged into small intestine, fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart), or administered orally by a enterically coated capsule for mouse consumption, e.g. capsule from example 32. Mice are killed by CO2 inhalation 8 h or 4 d later and whole blood is obtained by cardiac puncture.
Whole blood samples from above are added to e.g. ReticOne Reagent according to the manufacturer's instructions. Flow cytometry is performed with e.g. a Coulter Epics XL machine. Acquisition parameters are calibrated each time by e.g. Retic-Cal Biological Calibration and Retic-C Cell Control. 40,000 total events in the red blood cell gate are acquired and analyzed with ReticOne automated software for percentage of reticulocytes (all materials e.g. from Beckman Coulter). Increase in number of reticulocytes in blood is indicative of functional delivery of Epo-R agonists into body, i.e. systemic delivery.
Note: For Epo-R dimerization, the agonistically acting Nanobody construct may have a Koff equal or lower than 1 nM since interaction of the first site of Epo for EpoR is of high affinity (dissociation constant 1 nM) while the second binding interaction is much weaker (1 uM). To be determined e.g. in BioCore experiments.
Similarly as above, 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 Gly linker), optionally comprising a pH independent or pH dependent anti-mouse FcRn or pIgR Nanobody (i.e. binding at gut pH, about pH 6, but released at blood pH7 or more) are a) injected intraperitoneally, b) gauged into small intestine, c) fed intragastrically using a ball-point needle (once, twice, or four times 12 h apart), or d) administered orally by e.g. a enterically coated capsule acceptable for mouse consumption, e.g. capsule from example 32. Mice growth is monitored.
Increase in growth is indicative of a systemically delivered GHR agonist.
Note: For GHR, the agonistically acting Nanobody construct against GHR from above may have a Koff equal or lower than 0.3 nM since interaction of GH to GHR-dimer was reported to be in the range of 0.3 nM (Cunningham et al, 1989, Science 244:1081-1085.). To be determined e.g. in BioCore experiments.
Number | Date | Country | Kind |
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60875990 | Dec 2006 | US | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP07/64344 | 12/20/2001 | WO | 00 | 1/1/2010 |