ANTI-SERUM ALBUMIN BIDING VARIABLE DOMAINS
The invention relates to improved anti-serum albumin immunoglobulin single variable domains, as well as ligands and drug conjugates comprising such variable domains, compositions, nucleic acids, vectors and hosts.
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This application is a continuation of U.S. application Ser. No. 13/384,316, filed Jan. 16, 2012, which is a 371 of International Application No. PCT/EP2010/060112, filed 14 Jul. 2010, which claims the benefit of U.S. Provisional Application Nos. 61/226,028, filed 16 Jul. 2009 and 61/307,554 filed 24 Feb. 2010, which are incorporated by reference in their entireties.
The invention relates to improved anti-serum albumin immunoglobulin single variable domains, as well as ligands and drug conjugates comprising such domains, compositions, nucleic acids, vectors and hosts.
BACKGROUND OF THE INVENTIONWO04003019 and WO2008/096158 disclose anti-serum albumin (SA) binding moieties, such as anti-SA immunoglobulin single variable domains (dAbs), which have therapeutically-useful half-lives. These documents disclose monomer anti-SA dAbs as well as multi-specific ligands comprising such dAbs, eg, ligands comprising an anti-SA dAb and a dAb that specifically binds a target antigen, such as TNFR1. Binding moieties are disclosed that specifically bind serum albumins from more than one species, eg human/mouse cross-reactive anti-SA dAbs.
WO05118642 and WO2006/059106 disclose the concept of conjugating or associating an anti-SA binding moiety, such as an anti-SA immunoglobulin single variable domain, to a drug, in order to increase the half-life of the drug. Protein, peptide and NCE (chemical entity) drugs are disclosed and exemplified. WO2006/059106 discloses the use of this concept to increase the half-life of insulintropic agents, eg, incretin hormones such as glucagon-like peptide (GLP)-1.
Reference is also made to Holt et al, “Anti-Serum albumin domain antibodies for extending the half-lives of short lived drugs”, Protein Engineering, Design & Selection, vol 21, no 5, pp 283-288, 2008.
It would be desirable to provide improved heavy chain variable domain dAbs that specifically bind serum albumin, preferably albumins from human and non-human species, which would provide utility in animal models of disease as well as for human therapy and/or diagnosis. It would also be desirable to provide for the choice between relatively modest- and high-affinity anti-SA binding moieties (dAbs). Such moieties could be linked to drugs, the anti-SA binding moiety being chosen according to the contemplated end-application. This would allow the drug to be better tailored to treating and/or preventing chronic or acute indications, depending upon the choice of anti-SA binding moiety. It would also be desirable to provide anti-SA dAbs that are monomeric or substantially so in solution. This would especially be advantageous when the anti-SA dAb is linked to a binding moiety, eg, a dAb, that specifically binds a cell-surface receptor, such as TNFR1, with the aim of antagonizing the receptor. The monomeric state of the anti-SA dAb is useful in reducing the chance of receptor cross-linking, since multimers are less likely to form which could bind and cross-link receptors (eg, TNFR1) on the cell surface, thus increasing the likelihood of receptor agonism and detrimental receptor signaling. It would also be desirable to provide anti-SA dAbs that have relatively high melting temperatures. This is useful for providing stable formulations, eg, storage-stable formulations and variable domains that have a good shelf-life.
SUMMARY OF THE INVENTIONAspects of the present invention solve these problems.
In one aspect the invention, therefore, there is provided an anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 97 to 191 and 198 to 203.
An aspect of the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence having up to 4 amino acid changes compared to an amino acid sequence selected from SEQ ID NOs: 97 to 191 and 198 to 203.
An aspect of the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence that is encoded by a nucleotide sequence which is at least 80% identical to a sequence selected from SEQ ID NOs 1 to 96 and 192-197.
An aspect of the invention provides a multispecific ligand comprising an anti-SA variable domain of the invention and a binding moiety that specifically binds a target antigen other than SA.
An aspect of the invention provides an anti-SA single variable domain of the invention, wherein the variable domain is conjugated to a drug (optionally an NCE drug).
An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or NCE (new chemical entity) drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated (for an NCE) to any anti-SA variable domain of the invention. For example, the variable domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203 (or an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203).
An aspect of the invention provides a composition comprising a variable domain, fusion protein or ligand of the invention and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variable domain, or a multispecific ligand or fusion protein of the invention.
An aspect of the invention provides a nucleic acid comprising a nucleotide sequence that is at least 80% identical to a sequence selected from SEQ ID NOs 1 to 96 and 192-197.
An aspect of the invention provides a vector comprising the nucleic acid of the invention.
An aspect of the invention provides an isolated host cell comprising the vector of the invention.
An aspect of the invention provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variable domain, or a multispecific ligand or fusion protein of the invention to said patient.
Embodiments of any aspect of the invention provide anti-serum albumin single variable domains of good anti-serum albumin affinities. The choice of variable domain can allow for tailoring of half-life according to the desired therapeutic and/or prophylactic setting. For example, in one embodiment, the affinity of the variable domain for serum albumin is relatively high, such that the variable domain would be useful for inclusion in products that find utility in treating and/or preventing chronic or persistent diseases, conditions, toxicity or other chronic indications. In one embodiment, the affinity of the variable domain for serum albumin is relatively modest, such that the variable domain would be useful for inclusion in products that find utility in treating and/or preventing acute diseases, conditions, toxicity or other acute indications. In one embodiment, the affinity of the variable domain for serum albumin is intermediate, such that the variable domain would be useful for inclusion in products that find utility in treating and/or preventing acute or chronic diseases, conditions, toxicity or other acute or chronic indications.
It is conceivable that a molecule with an appropriately high affinity and specificity for serum albumin would stay in circulation long enough to have the desired therapeutic effect. (Tomlinson, Nature Biotechnology 22, 521-522 (2004)). Here, a high affinity anti-SA variable domain would stay in serum circulation matching that of the species' serum albumin (WO2008096158). Once in circulation, any fused therapeutic agent to the AlbudAb variable domain, be it NCE, peptide or protein, consequently would be able to act longer on its target and exhibit a longer lasting therapeutic effect. This would allow for targeting chronic or persisitent diseases without the need of frequent dosing.
A variable domain with moderate affinity, (but specificity to SA) would only stay in serum circulation for a short time (eg, for a few hours or a few days) allowing for the specific targeting of therapeutic targets involved in acute diseases by the fused therapeutic agent.
This way it is possible to tailor the anti-SA-containing product to the therapeutic disease area by choosing an anti-SA variable domain with the appropriate albumin binding affinity and/or serum half-life.
DETAILED DESCRIPTION OF THE INVENTIONWithin this specification the invention has been described, with reference to embodiments, in a way which enables a clear and concise specification to be written. It is intended and should be appreciated that embodiments may be variously combined or separated without parting from the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.
As used herein, the term “antagonist of Tumor Necrosis Factor Receptor 1 (TNFR1)” or “anti-TNFR1 antagonist” or the like refers to an agent (e.g., a molecule, a compound) which binds TNFR1 and can inhibit a (i.e., one or more) function of TNFR1. For example, an antagonist of TNFR1 can inhibit the binding of TNFα to TNFR1 and/or inhibit signal transduction mediated through TNFR1. Accordingly, TNFR1-mediated processes and cellular responses (e.g., TNFα-induced cell death in a standard L929 cytotoxicity assay) can be inhibited with an antagonist of TNFR1.
A “patient” is any animal, eg, a mammal, eg, a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the patient is a human.
As used herein, “peptide” refers to about two to about 50 amino acids that are joined together via peptide bonds.
As used herein, “polypeptide” refers to at least about 50 amino acids that are joined together by peptide bonds. Polypeptides generally comprise tertiary structure and fold into functional domains.
As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab, F(ab′)2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
As used herein, “antibody format” refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure. A variety of suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment), a single antibody variable domain (e.g., a dAb, VH, VHH, VL), and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized VHH).
The phrase “immunoglobulin single variable domain” refers to an antibody variable domain (VH, VHH, VL) that specifically binds an antigen or epitope independently of different V regions or domains. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single immunoglobulin variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single antibody variable domain” or an “antibody single variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. The VHH may be humanized.
A “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. A “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
In the instant application, the term “prevention” and “preventing” involves administration of the protective composition prior to the induction of the disease or condition. “Treatment” and “treating” involves administration of the protective composition after disease or condition symptoms become manifest. “Suppression” or “suppressing” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease or condition.
As used herein, the term “dose” refers to the quantity of ligand administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval. For example, dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single variable domain that binds target antigen) administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g., by a single administration, or by two or more administrations). The interval between doses can be any desired amount of time. The term “pharmaceutically effective” when referring to a dose means sufficient amount of the ligand, domain or pharmaceutically active agent to provide the desired effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent and the like. Thus, it is not always possible to specify an exact “effective” amount applicable for all patients. However, an appropriate “effective” dose in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
Methods for pharmacokinetic analysis and determination of ligand (eg, single variable domain, fusion protein or multi-specific ligand) half-life will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC). Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a human. Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a mouse or rat or Cynomolgus monkey.
Half lives (t½ alpha and t½ beta) and AUC can be determined from a curve of serum concentration of ligand against time. The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve. When two-compartment modeling is used, in a first phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some elimination. A second phase (beta phase) is the phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase. Thus, in one embodiment, in the context of the present invention, the variable domain, fusion protein or ligand has a tα half-life in the range of (or of about) 15 minutes or more. In one embodiment, the lower end of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention will have a ta half life in the range of up to and including 12 hours (or about 12 hours). In one embodiment, the upper end of the range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
In one embodiment, the present invention provides the variable domain, fusion protein or ligand according to the invention has a tβ half-life in the range of (or of about) 2.5 hours or more. In one embodiment, the lower end of the range is (or is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In addition, or alternatively, the tβ half-life is (or is about) up to and including 21 or 25 days. In one embodiment, the upper end of the range is (or is about) 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days 20 days, 21 days or 22 days. For example, the variable domain, fusion protein or ligand according to the invention will have a tβ half life in the range 12 to 60 hours (or about 12 to 60 hours). In a further embodiment, it will be in the range 12 to 48 hours (or about 12 to 48 hours). In a further embodiment still, it will be in the range 12 to 26 hours (or about 12 to 26 hours).
As an alternative to using two-compartment modeling, the skilled person will be familiar with the use of non-compartmental modeling, which can be used to determine terminal half-lives (in this respect, the term “terminal half-life” as used herein means a terminal half-life determined using non-compartmental modeling). The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve in this way. In this instance, in one embodiment the single variable domain, fusion protein or ligand has a terminal half life of at least (or at least about) 8 hours, 10 hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In one embodiment, the upper end of this range is (or is about) 24 hours, 48 hours, 60 hours or 72 hours or 120 hours. For example, the terminal half-life is (or is about) from 8 hours to 60 hours, or 8 hours to 48 hours or 12 to 120 hours, eg, in man.
In addition, or alternatively to the above criteria, the variable domain, fusion protein or ligand according to the invention has an AUC value (area under the curve) in the range of (or of about) 1 mg.min/ml or more. In one embodiment, the lower end of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300 mg.min/ml. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention has an AUC in the range of (or of about) up to 600 mg.min/ml. In one embodiment, the upper end of the range is (or is about) 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously the variable domain, fusion protein or ligand will have an AUC in (or about in) the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50 mg.min/ml.
“Surface Plasmon Resonance”: Competition assays can be used to determine if a specific antigen or epitope, such as human serum albumin, competes with another antigen or epitope, such as cynomolgus serum albumin, for binding to a serum albumin binding ligand described herein, such as a specific dAb. Similarly competition assays can be used to determine if a first ligand such as dAb, competes with a second ligand such as a dAb for binding to a target antigen or epitope. The term “competes” as used herein refers to substance, such as a molecule, compound, preferably a protein, which is able to interfere to any extent with the specific binding interaction between two or more molecules. The phrase “does not competitively inhibit” means that substance, such as a molecule, compound, preferably a protein, does not interfere to any measurable or significant extent with the specific binding interaction between two or more molecules. The specific binding interaction between two or more molecules preferably includes the specific binding interaction between a single variable domain and its cognate partner or target. The interfering or competing molecule can be another single variable domain or it can be a molecule that that is structurally and/or functionally similar to a cognate partner or target.
The term “binding moiety” refers to a domain that specifically binds an antigen or epitope independently of a different epitope or antigen binding domain. A binding moiety may be a domain antibody (dAb) or may be a domain which is a derivative of a non-immunoglobulin protein scaffold, eg, a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, an adnectin, affibody, an avimer, GroEl, transferrin, GroES and fibronectin, which binds to a ligand other than the natural ligand (in the case of the present invention, the moiety binds serum albumin). See WO2008/096158, which discloses examples of protein scaffolds and methods for selecting antigen or epitope-specific binding domains from repertoires (see Examples 17 to 25). These specific disclosures of WO2008/096158 are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
In one embodiment, a variable domain of the invention comprises one or more of the following kinetic characteristics:—
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- (a) The variable domain comprises a binding site that specifically binds human SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (b) The variable domain comprises a binding site that specifically binds human SA with an off-rate constant (Kd) from (or from about) 1.5×10−4 to (or to about) 0.1 sec−1, optionally from (or from about) 3×10−4 to (or to about) 0.1 sec as determined by surface plasmon resonance;
- (c) The variable domain comprises a binding site that specifically binds human SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104 M−1sec−1, optionally from (or from about) 1×106 to (or to about) 2×104 M−1sec−1 as determined by surface plasmon resonance;
- (d) The variable domain comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (e) The variable domain of any preceding claim, wherein the variable domain comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (Kd) from (or from about) 1.5×10−4 to (or to about) 0.1 sec−1, optionally from (or from about) 3×10−4 to (or to about) 0.1 sec−1 as determined by surface plasmon resonance;
- (f) The variable domain of any preceding claim, wherein the variable domain comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104M−1sec−1, optionally from (or from about) 1×106 to (or to about) 5×103 M−1sec−1 as determined by surface plasmon resonance;
- (g) The variable domain comprises a binding site that specifically binds rat SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM, optionally from (or from about) 20 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (h) The variable domain comprises a binding site that specifically binds rat SA with an off-rate constant (Ka) from (or from about) 2×10−3 to (or to about) 0.15 sec−1, optionally from (or from about) 9×10−3 to (or to about) 0.14 sec−1 as determined by surface plasmon resonance;
- (i) The variable domain comprises a binding site that specifically binds rat SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104M−1sec−1, optionally from (or from about) 1×106 to (or to about) 3×104 M−1sec−1 as determined by surface plasmon resonance;
- (j) The variable domain comprises a binding site that specifically binds mouse SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM as determined by surface plasmon resonance;
- (k) The variable domain comprises a binding site that specifically binds mouse SA with an off-rate constant (Kd) from (or from about) 2×10−3 to (or to about) 0.15 sec−1 as determined by surface plasmon resonance; and/or
- (l) The variable domain comprises a binding site that specifically binds mouse SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104 M−1sec−1, optionally from (or from about) 2×106 to (or to about) 1.5×104 M−1sec−1 as determined by surface plasmon resonance.
Optionally, the variable domain has
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- I: a KD according to (a) and (d), a Kd according to (b) and (e), and a Ka according to (c) and (f); or
- II: a KD according to (a) and (g), a Kd according to (b) and (h), and a Ka according to (c) and (i); or
- III: a KD according to (a) and (j), a Kd according to (b) and (k), and a Ka according to (c) and (l); or
- IV. kinetics according to I and II; or
- V: kinetics according to I and III; or
- VI: kinetics according to I, II and III.
The invention also provides a ligand comprising a variable domain of any preceding aspect or embodiment of the invention. For example, the ligand can be a dual-specific ligand (see WO04003019 for examples of dual-specific ligands). In one aspect, the invention provides a multispecific ligand comprising an anti-SA variable domain of any preceding aspect or embodiment of the invention and a binding moiety that specifically binds a target antigen other than SA. The binding moiety can be any binding moiety that specifically binds a target, eg, the moiety is an antibody, antibody fragment, scFv, Fab, dAb or a binding moiety comprising a non-immunoglobulin protein scaffold. Such moieties are disclosed in detail in WO2008/096158 (see examples 17 to 25, which disclosure is incorporated herein by reference). Examples of non-immunoglobulin scaffolds are CTLA-4, lipocallin, staphylococcal protein A (spA), Affibody™, Avimers™, adnectins, GroEL and fibronectin.
In one embodiment, a linker is provided between the anti-target binding moiety and the anti-SA variable domain, the linker comprising the amino acid sequence AST, optionally ASTSGPS. Alternative linkers are described in WO2007085814 (incorporated herein by reference) and WO2008/096158 (see the passage at page 135, line 12 to page 140, line 14, which disclosure and all sequences of linkers are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
In one embodiment of the multispecific ligand, the target antigen may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic. In this respect, the ligand of the invention may bind the target antigen and act as an antagonist or agonist (e.g., EPO receptor agonist). One skilled in the art will appreciate that the choice is large and varied. They may be for instance, human or animal proteins, cytokines, cytokine receptors, where cytokine receptors include receptors for cytokines, enzymes, co-factors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include, but are preferably not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-131, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5, non-structural protein type 3 (NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12, internalizing receptors that are over-expressed on certain cells, such as the epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor cells, an internalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular matrix protein, elastin, fibronectin, laminin, a 1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and an antigen of influenza virus. It will be appreciated that this list is by no means exhaustive.
In one embodiment, the multispecific ligand comprises an anti-SA dAb variable domain of the invention and an anti-TNFR1 binding moiety, e.g., an anti-TNFR1 dAb. Optionally, the ligand has only one anti-TNFR1 binding moiety (e.g., dAb) to reduce the chance of receptor cross-linking. In one embodiment, the anti-SA dAb comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203 (or an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203).
In one embodiment, the anti-TNFR1 binding moiety is DOM1h-131-206 disclosed in WO2008149148 (the amino acid sequence of which and the nucleotide sequence of which, as disclosed in that PCT application, are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein). In one embodiment, the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-131-206 and the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203 (or an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203).
In one embodiment, the anti-TNFR1 binding moiety or dAb is any such moiety or dAb disclosed in co-pending application U.S. Ser. No. 61/153,746, the disclosure of which is incorporated herein by reference. In one embodiment, the anti-TNFR1 binding moiety comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of DOM1h-574-156, DOM1h-574-72, DOM1h-574-109, DOM1h-574-138, DOM1h-574-162 or DOM1h-574-180 or the amino acid sequence of any anti-TNFR1 dAb disclosed herein. In one embodiment, the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-574-156 and the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203 (or an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203).
In one embodiment, the ligand of the invention is a fusion protein comprising a variable domain of the invention fused directly or indirectly to one or more polypeptides. For example, the fusion protein can be a “drug fusion” as disclosed in WO2005/118642 (the disclosure of which is incorporated herein by reference), comprising a variable domain of the invention and a polypeptide drug as defined in that PCT application.
As used herein, “drug” refers to any compound (e.g., small organic molecule, nucleic acid, polypeptide) that can be administered to an individual to produce a beneficial, therapeutic or diagnostic effect through binding to and/or altering the function of a biological target molecule in the individual. The target molecule can be an endogenous target molecule encoded by the individual's genome (e.g. an enzyme, receptor, growth factor, cytokine encoded by the individual's genome) or an exogenous target molecule encoded by the genome of a pathogen (e.g. an enzyme encoded by the genome of a virus, bacterium, fungus, nematode or other pathogen). Suitable drugs for use in fusion proteins and conjugates comprising an anti-SA dAb domain of the invention are disclosed in WO2005/118642 and WO2006/059106 (the entire disclosures of which are incorporated herein by reference, and including the entire list of specific drugs as though this list were expressly written herein, and it is contemplated that such incorporation provides disclosure of specific drugs for inclusion in claims herein). For example, the drug can be glucagon-like peptide 1 (GLP-1) or a variant, interferon alpha 2b or a variant or exendin-4 or a variant.
In one embodiment, the invention provides a drug conjugate as defined and disclosed in WO2005/118642 and WO2006/059106, wherein the conjugate comprises a variable domain of the invention. In one example, the drug is covalently linked to the variable domain (eg, the variable domain and the drug are expressed as part of a single polypeptide). Alternatively, in an example, the drug is non-covalently bonded or associated with the variable domain. The drug can be covalently or noncovalently bonded to the variable domain directly or indirectly (e.g., through a suitable linker and/or noncovalent binding of complementary binding partners (e.g., biotin and avidin)). When complementary binding partners are employed, one of the binding partners can be covalently bonded to the drug directly or through a suitable linker moiety, and the complementary binding partner can be covalently bonded to the variable domain directly or through a suitable linker moiety. When the drug is a polypeptide or peptide, the drug composition can be a fusion protein, wherein the polypeptide or peptide, drug and the polypeptide binding moiety are discrete parts (moieties) of a continuous polypeptide chain. As described herein, the polypeptide binding moieties and polypeptide drug moieties can be directly bonded to each other through a peptide bond, or linked through a suitable amino acid, or peptide or polypeptide linker.
A ligand which contains one single variable domain (eg, monomer) of the invention or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, can further comprise one or more entities selected from, but preferably not limited to a label, a tag, an additional single variable domain, a dAb, an antibody, and antibody fragment, a marker and a drug. One or more of these entities can be located at either the COOH terminus or at the N terminus or at both the N terminus and the COOH terminus of the ligand comprising the single variable domain, (either immunoglobulin or non-immunoglobulin single variable domain). One or more of these entities can be located at either the COOH terminus, or the N terminus, or both the N terminus and the COOH terminus of the single variable domain which specifically binds serum albumin of the ligand which contains one single variable domain (monomer) or more than one single variable domains (multimer, fusion protein, conjugate, and dual specific ligand as defined herein). Non-limiting examples of tags which can be positioned at one or both of these termini include a HA, his or a myc tag. The entities, including one or more tags, labels and drugs, can be bound to the ligand which contains one single variable domain (monomer) or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein), which binds serum albumin, either directly or through linkers as described above.
An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or conjugate with an NCE (new chemical entity) drug, comprising a polypeptide drug fused or conjugated (for an NCE) to any variable domain as described above, optionally wherein the variable domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203 (or an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203).
The invention provides a composition comprising a variable domain, fusion protein, conjugate or ligand of any aspect of the invention and a pharmaceutically acceptable diluent, carrier, exipient or vehicle.
Also encompassed herein is an isolated nucleic acid encoding any of the variable domain, fusion proteins, conjugates or ligands described herein, e.g., a ligand which contains one single variable domain (eg, monomer) of the invention or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, or which specifically binds both human serum albumin and at least one non-human serum albumin, or functionally active fragments thereof. Also encompassed herein is a vector and/or an expression vector, a host cell (eg, a non-human host cell or a host cell that is not isolated from a human or human embryo) comprising the vector, e.g., a plant or animal cell and/or cell line transformed with a vector, a method of expressing and/or producing one or more variable domains, fusion proteins or ligands which contains one single variable domain (monomer) or more than one single variable domains (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, or fragment(s) thereof encoded by said vectors, including in some instances culturing the host cell so that the one or more variable domains, fusion proteins or ligands or fragments thereof are expressed and optionally recovering the ligand which contains one single variable domain (monomer) or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, from the host cell culture medium. Also encompassed are methods of contacting a ligand described herein with serum albumin, including serum albumin and/or non-human serum albumin(s), and/or one or more targets other than serum albumin, where the targets include biologically active molecules, and include animal proteins, cytokines as listed above, and include methods where the contacting is in vitro as well as administering any of the variable domains, fusion proteins or ligands described herein to an individual host animal or cell in vivo and/or ex vivo. Preferably, administering ligands described herein which comprises a single variable domain (immunoglobulin or non-immunoglobulin) directed to serum albumin and/or non-human serum albumin(s), and one or more domains directed to one or more targets other than serum albumin, will increase the half life, including the T beta and/or terminal half life, of the anti-target ligand. Nucleic acid molecules encoding the domains, fusion proteins or single domain containing ligands or fragments thereof, including functional fragments thereof, are contemplated herein. Vectors encoding the nucleic acid molecules, including but preferably not limited to expression vectors, are contemplated herein, as are host cells from a cell line or organism containing one or more of these expression vectors. Also contemplated are methods of producing any domain, fusion protein or ligand, including, but preferably not limited to any of the aforementioned nucleic acids, vectors and host cells.
An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variable domain according to the invention or a multispecific ligand of the invention or fusion protein of the invention.
An aspect of the invention provides a nucleic acid comprising the nucleotide sequence selected from any one of SEQ ID NOs: 1 to 96 and 192-197, or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said selected sequence.
An aspect of the invention provides a vector comprising the nucleic acid of the invention. An aspect of the invention provides an isolated host cell comprising the vector.
Reference is made to WO2008/096158 for details of library vector systems, combining single variable domains, characterization of dual specific ligands, structure of dual specific ligands, scaffolds for use in constructing dual specific ligands, uses of anti-serum albumin dAbs and multispecific ligands and half-life-enhanced ligands, and compositions and formulations of comprising anti-serum albumin dAbs. These disclosures are incorporated herein by reference to provide guidance for use with the present invention, including for domains, ligands, fusion proteins, conjugates, nucleic acids, vectors, hosts and compositions of the present invention.
The routine bacterial expression level in 2.5 L shake flasks was determined following culture in Onex media at 30° C. for 48 hrs at 250 rpm. The biophysical characteristics were determined by SEC MALLS and DSC.
SEC MALLS (size exclusion chromatography with multi-angle-LASER-light-scattering) is a non-invasive technique for the characterizing of macromolecules in solution. Briefly, proteins (at concentration of 1 mg/mL in buffer Dulbecco's PBS) are separated according to their hydrodynamic properties by size exclusion chromatography (column: TSK3000; S200). Following separation, the propensity of the protein to scatter light is measured using a multi-angle-LASER-light-scattering (MALLS) detector. The intensity of the scattered light while protein passes through the detector is measured as a function of angle. This measurement taken together with the protein concentration determined using the refractive index (R1) detector allows calculation of the molar mass using appropriate equations (integral part of the analysis software Astra v.5.3.4.12). The highest concentration at the mid-point of the eluting peak is about 8-10 uM and this consequently is the concentration at which MALLS determines the in-solution state of the protein.
DSC (Differential Scanning calorimetry): briefly, the protein is heated at a constant rate of 180 degrees C./hrs (at 1 mg/mL in PBS) and a detectable heat change associated with thermal denaturation measured. The transition midpoint (appTm) is determined, which is described as the temperature where 50% of the protein is in its native conformation and the other 50% is denatured. Here, DSC determined the apparent transition midpoint (appTm) as most of the proteins examined do not fully refold. The higher the Tm, the more stable the molecule. The software package used was OriginR v7.0383.
Characteristics of the VH dAbs are summarised in Table 1 below. Cross-reactivity of the AlbudAbs™ (i.e., anti-serum albumin dAbs) was determined against human, Cynomolgus monkey (cyno), rat and mouse serum albumin (“4AGs” in the table) using surface plasmon resonance (SPR). In this case, Biacore™ was used. The epitope mapping to domain 1, 2 and/or 3 (D1,2,3) of human serum albumin (HSA) was performed using SPR and purified individual domains of HSA (in-house) covalently coupled to a CM5 chip (amine coupling). The expression was in 2.5 L baffled glass flasks in a volume of 500 mL in OverNight Express™ at 30 C, 250 rpm.
MALLS results: A single VH AlbudAb is 14 kDa in size. Any value between 14 and 28 kDa as determined by MALLS is indicative of varying degrees of self-association or dimer formation (i.e. 16 kDa predominately monomeric under the conditions tested whereas 22 kDa indicates a strong propensity to dimerise under MALLS conditions).
DSC results: The concentration of protein in a DSC experiment is much higher at 1 mg/mL in the actual reaction cell compared to MALLS. This higher concentration could explain in part the presence of two appTms for some AlbudAbs as seen in tablel; the first Tm constitutes the dissociation of the dimeric complex, whereas the second Tm represents the unfolding of the actual AlbudAb protein.
Apart from DOM7h-112, all above AlbudAbs leads are fully cross-reactive between the four species of serum albumin. All identified AlbudAbs bind Domain2 of HSA and express reasonably well in shake flasks under non-optimised conditions. 5 out of 7 AlbudAbs are monomeric as determined by MALLS, whereas DOM7r-35 shows a significant propensity to dimerise under the MALLS conditions. Monomeric state is advantageous because it avoids dimerisation and the risk of products that may cross-link targets such as cell-surface receptors.
DOM7r-36 shows some degree of aggregate formation (less than 10% when quantified on MALLS). For 5 out of 7 AlbudAbs, 2 appTms can be determined. This is due to the higher experimental concentration in DSC experiments and slightly different in-solution state of the dAb at this elevated concentration (for details, see explanation also above).
Example 2 Determination of Serum Half Life in RatAlbudAbs were cloned into the pDOM5 vector. The pDOM5 vector is a pUC 119-based expression vector where protein expression is driven by the LacZ promoter. A GAS 1 leader sequence (see WO 2005/093074) ensures secretion of isolated, soluble dAbs into the periplasm and culture supernatant of E. coli. dAbs are cloned SalI/NotI in this vector, which appends a myc tag at the C-terminus of the dAb. For each AlbudAb, 20-50 mg quantities were expressed in E. coli and purified from bacterial culture supernatant using protein A affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience). For Rat pharmacokinetic (PK) analysis, AlbudAbs were dosed as single i.v injections at 2.5 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 12, 24, 48, 72, 96, 120, 168 hrs. Analysis of serum levels was by anti-myc capture followed by anti-VH detection ELISA as per the method described below.
Results are shown in table 2. All tested AlbudAbs show a serum-half life extending ability (negative control HEL4 dAb with T1/2 of 20mins in rat) to varying degrees; this trend can also be seen in the calculated AUC being the highest value for the longest t1/2. The longest serum half-life with 34.5 hrs approximates the serum half-life of rat serm albumin.
The specific affinities of the AlbudAbs to RSA will need to be determined.
The AlbudAb concentration in serum was measured by anti-myc ELISA. Briefly, goat anti-myc polyclonal antibody (1:500; Abcam, catalogue number ab9132) was coated overnight onto Nunc 96-well Maxisorp plates and blocked with 5% BSA/PBS+1% TWEEN™. Serum samples were added at a range of dilutions alongside a standard at known concentrations. Bound myc-tagged AlbudAb was then detected using a rabbit polyclonal anti-VH directly labelled with the MSD sulfo-tag. Each dAb was diluted in assay buffer containing 10% control rat serum (1:1000; in-house reagent) (method DM222). MSD (MesoScaleDiscovery; MesoScale.com) utilizes electrochemiluminescence detection of the sulfo-tag after electrochemical stimulus.
From the raw ELISA data, the concentration of unknown samples was established by interpolation against the standard curve taking into account dilution factors. The mean concentration result from each time point was determined from replicate values and entered into WinNonLin analysis package (eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA). The data was fitted using a non-compartmental model, where PK parameters were estimated by the software to give terminal half-lives. Dosing information and time points were selected to reflect the terminal phase of each PK profile.
Example 3 Affinity Maturation of Naïve VH AlbudAbs™12 VH AlbudAb leads isolated from naïve selection were taken forward for affinity maturation. Individual error prone libraries (EP) of DOM7r-36, DOM7r-35, DOM7r-31, DOM7h-98, DOM7h-112, DOM7r-38 and DOM7r-29 were made, whereas the following parental clones were pooled and combined in a single EP library and screened together: DOM7r-83, DOM7r-85, DOM7r-92, DOM7r-94 and DOM7r-95. All libraries were greater than 2×109 CFU/mL.
Selections were performed in 4 rounds on soluble antigen (biotin-HSA; biotin-RSA; blocking with 2% Marvel) by cross over-selection with decreasing concentration of antigen: Round1 at 1 μM (HSA or RSA), Round 2 at 1 μM (RSA or HSA), followed by 2 further rounds of selection at 100 nM and 10 nM, respectively, with the same antigen as in Round2. Ca. 3000 samples from both, R3 and R4 outputs were screened by supernatant BIAcore and clones ranked according to their off-rate only. Eight-point dilution kinetic affinity measurements were performed on improved clones (data below).
The CDRs are underlined; sequences are shown N- to C-terminus; “˜” denote gaps introduced for alignment
Claims
1. An anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 97 to 191 and 198 to 203.
2. An anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence having up to 4 amino acid changes compared to an amino acid sequence selected from SEQ ID NOs: 97 to 191 and 198 to 203.
3. An anti-serum albumin (SA) immunoglobulin single variable domain comprising an amino acid sequence that is encoded by a nucleotide sequence which is at least 80% identical to a sequence selected from SEQ ID NOs 1 to 96 and 192 to 197.
4. The variable domain of claim 1, wherein the variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 97 to 103 and 198 to 203.
5. The variable domain of claim 1, comprising a binding site that specifically binds human SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance.
6. The variable domain of claim 1, comprising a binding site that specifically binds human SA with an off-rate constant (Kd) of from about 1.5×10−4 to about 0.1 sec−1, optionally from about 3×10−4 to about 0.1 sec as determined by surface plasmon resonance.
7. The variable domain of claim 1, comprising a binding site that specifically binds human SA with an on-rate constant (Ka) of from about 2×106 to about 1×104 M−1sec−1, optionally from about 1×106 to about 2×104M−1sec−1 as determined by surface plasmon resonance.
8. The variable domain of claim 1, comprising a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance.
9. The variable domain of claim 1, comprising a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (Kd) of from about 1.5×10−4 to about 0.1 sec−1, optionally from about 3×10−4 to about 0.1 sec−1 as determined by surface plasmon resonance.
10. The variable domain of claim 1, comprising a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (Ka) of from about 2×106 to about 1×104M−1sec−1, optionally from about 1×106 to about 5×103M−1sec−1 as determined by surface plasmon resonance.
11. The variable domain of claim 1, wherein the variable domain has a melting temperature (Tm) of at least 55 degrees centigrade, optionally 55≦Tm≦75 degrees centigrade, as determined by DSC (differential scanning calorimetry).
12. The variable domain of claim 1, wherein the variable domain is substantially monomeric as determined by SEC-MALLS (size exclusion chromatography with multi-angle-LASER-light-scattering).
13. A multispecific ligand comprising an anti-SA variable domain of claim 1 and a binding moiety that specifically binds a target antigen other than SA, optionally wherein the binding moiety is an TNFR1 antagonist.
14. An anti-SA single variable domain of claim 1, wherein the variable domain is conjugated to a drug (optionally an NCE drug).
15. A fusion protein comprising a polypeptide or peptide drug fused to a variable domain according to claim 1.
16. A composition comprising a variable domain, fusion protein or ligand of claim 1 and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
17. A nucleic acid comprising a nucleotide sequence encoding a variable domain according to claim 1.
18. A vector comprising the nucleic acid of claim 17.
19. An isolated host cell comprising the vector of claim 18.
20. A method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variable domain according to claim 1.
Type: Application
Filed: Jan 30, 2014
Publication Date: May 22, 2014
Applicant: Glaxo Group Limited (Greenford)
Inventors: Edward COULSTOCK (Cambridge), Elena De Angelis (Cambridge), Haiqun Liu (Cambridge), Oliver Schon (Cambridge)
Application Number: 14/168,168
International Classification: C07K 16/18 (20060101);
