WO2013093465A2 - Nouvelles formulations - Google Patents

Nouvelles formulations Download PDF

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Publication number
WO2013093465A2
WO2013093465A2 PCT/GB2012/053195 GB2012053195W WO2013093465A2 WO 2013093465 A2 WO2013093465 A2 WO 2013093465A2 GB 2012053195 W GB2012053195 W GB 2012053195W WO 2013093465 A2 WO2013093465 A2 WO 2013093465A2
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composition according
disease
antibody
group
acute
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WO2013093465A3 (fr
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Jonas Heilskov Graversen
Gabriele Anton
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CYTOGUIDE APS
SMITH STEPHEN E
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CYTOGUIDE APS
SMITH STEPHEN E
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6445—Haemoglobin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to the formulation of therapeutic agents for the targeted delivery of a therapeutic or diagnostic agent (or agents) covalently linked to a targeting moiety by an ester bond.
  • a key issue in the manufacturing of a protein-drug conjugates is to develop formulations that enable liquid storage of the drug for several months prior to its use, i.e., to increase shelf-life.
  • Protein conjugate drugs are used for the targeted delivery of a range of small molecules to specific cells of interest, where the small molecule should elicit its activity. This is achieved by linking the active drug, typically a small molecule known to have pharmaceutical activity, to a targeting protein agent with affinity for the target tissue or target cells. After binding to the target, the small molecule should be released, either after internalization into the target cell or by degradation on the cell surface.
  • linkers for conjugating drugs to proteins consist of two parts, a first part linking the drug to the protein and a second part constituting a biodegradable bond between drug and linker.
  • the link is most often made to primary amino groups, carbohydrate groups of the protein (after derivatisation to generate reactive groups), or to free sulfhydryl groups, generated by partial reduction of the protein.
  • the second part of the linker should be selectively degraded upon internalization so that active drug is liberated (and be sufficiently stable before internalization).
  • the degradable part of the linker is designed to be broken down by agents in the local environment where the drug is targeted. This can be facilitated by specific local enzymes, specific local pH (low or high) or a local reducing environment.
  • Ester linkages are commonly used as the biodegradable part of the linker to the protein. These esters are specifically hydrolyzed by esterases that are expressed to a higher level inside the cell than outside the cell. These esterases are expressed at an even higher level in the endosomes and lysosomes of a cell.
  • a minute amount of esterase may be released from a damaged, cancerous or inflamed cell leading to a higher esterase concentration near the cell surface than in the plasma, leading to local release of drug out-side the cell but near the site of interest.
  • Protein-conjugated drugs with ester linkers are known to be broken down not only by esterases but also by acidic or alkaline conditions. Hence, protein-conjugated drugs are formulated at neutral pH.
  • buffers with pH 4 or below have been used to hydrolyze the ester bonds and release drugs (see below).
  • Doxorubicin has been linked to a delivery polypeptide using an ester bond and tested at neutral pH (Meyer-Losic F., J. Med. Chem. (2006), 49, 6908-6916), no liquid formulation of the drug was made.
  • the cancer drug Paclitaxel (Taxol) has been conjugated to monoclonal antibodies (MC192 and 5C3) through a biodegradable ester linkage (Guillemard V et al, Cancer Res. (2001), 61 , 694-699).
  • the conjugate drug was formulated in PBS pH 7.4, and stored frozen.
  • the ester linkage is specifically described as low pH sensitive. Drug is released from the mAb by incubating at pH 4.0 for 48 hours.
  • dexamethasone-succinate also called dexamethasone-hemisuccinate, covalently linked to either serum albumin or a monoclonal antibody.
  • Dexamethasone conjugated through an ester linkage as succinate-dexamethasone to the amino groups of a monoclonal E-selectin antibody was formulated at neutral pH but stored frozen at -20°C. The antibody was frozen to increase stability, no studies were conducted or reported to evaluate if a non-frozen formulation could be developed (Everts et al, Journal of Immunology, (2002), 168, 883-889 and Everts M et al, Pharmaceutical research (2003), 20, 64-72), Kok, R.J. et al, Pharmaceutical research, (2002), 19, 1730- 1735 & Asgeirdottir SA, Biochem. Pharmacol. (2993), 1729-1739).
  • Albumin conjugated with dexamethasone-hemisuccinate has been prepared in a lyophilized form and the ester bond between dexamethasone and the linker is identified as the easiest to hydrolyze.
  • the stability of the drug has been investigated in PBS buffer and plasma and was described as comparable with each other and with a release below 10% after 24 hours (Melgert, B. N. et al, Journal of hepatology (2000), 32, 603-611). This release rate may be acceptable after injection, but for storage purposes 10% degradation per day of formulated drug on the shelf is not acceptable.
  • Triclosan linked to a polymer only studied the stability in neutral PBS, and reported relative fast release (Samuel B.U. et al, PNAS, (2003), 100(24), 14281-286).
  • a study examining the effect on length of linker between drug conjugated to a polymer by an ester made detailed studies on the linker and temperature effect on drug stability, recognizing that low in vitro conjugate stability was an issue, but ran all studies at pH 7.4 (Schenmakers RG, Jour Controlled release, (2004) 95, 291-300).
  • Dexamethasone linked to a dendrimer structure through an ester linkage has been reported (Navath, R. S. et al, Biomacromolecules (2010), 11 , 1544-1563). This was said to allow slower release of the drug, since dexamethasone was buried in a hydrogel formed by the dendrimer scaffold stabilizing the conjugate by shielding it. Dexamethasone was not released in vitro, since the hydrogel was not degraded when stored in vitro at neutral pH.
  • Methylprednisolone has been reported conjugated to dextran molecules of 25 kDa and shown to have a slower release rate at pH 4 in 50 mM acetate buffer, than at neutral pH (Penugonda S. et al., J. Pharm. Sci (2008), 97, 2649-2664). Also here it was demonstrated that local electronic environment near the drug was of importance for the release rate. Dextran conjugation is to a certain degree shielding a drug from its solvent, so the observed effect may also be due to differences in shielding efficiencies at different pH values. Penugonda et al., does not report if this effect can be expected for methylprednisolone conjugated to proteins or other entities, where the local electronic environment is different.
  • conjugation will most often be to primary amino groups of the lysine side-chain, and not to the amino group of a glycine in a very short peptide (2-5 residues) as in the paper.
  • the flexibility of a synthetic polymer is substantially larger than that for a protein with a well defined three dimensional structure, and higher degree of shielding of the drug entity from the solvent by the synthetic polymer may thus be expected. Disregarding the shielding effect of dextran.the conjugation chemistry in the paper corresponds to conjugation of an N- terminal amino group with a glycine as the N-terminal amino acid residue,.
  • the present invention describes how the stability of therapeutic agents for drug targeting containing a biodegradable ester bond can be formulated in a liquid form that is suitable for mid- to long-term storage, making the drugs easier to use and handle, and additionally lowering the cost of goods by increasing shelf life. Increased stability of the conjugate also leads to a more predictable effect of a defined drug dose.
  • the present invention teaches how to increase the stability of protein conjugate drugs with a degradable ester link between a small molecule drug and a protein targeting agent by formulating it at a mildly acidic pH.
  • a substantial decrease in release rate of drug from the conjugate can be achieved by making a slightly acidic formulation of the conjugate.
  • the therapeutic agent is a glucocorticoid, but any other therapeutic agent may equally be used.
  • the increased stability at slightly acidic pH is contrary to the general perception in the field, that ester bonds in protein conjugate drugs are sensitive to degradation at mildly acidic pH.
  • a first aspect of the invention provides a storage-stable composition
  • a storage-stable composition comprising a polypeptide binding moiety and a therapeutic agent (i.e., a protein-agent conjugate), wherein the polypeptide binding moiety is conjugated to the therapeutic agent by an ester bond and wherein the composition has an acidic pH.
  • a therapeutic agent i.e., a protein-agent conjugate
  • compositions comprising a protein-agent conjugate, directly or indirectly conjugated with an ester bond wherein (A) less than 5% per month of the therapeutic agent is released from the conjugate, and (B) less than 5% per month of the polypeptide binding moiety is denatured; when the composition is stored at standard room temperature (e.g., between 20°C and 25°C, and preferably 22 °C) in the absence of direct sunlight.
  • standard room temperature e.g., between 20°C and 25°C, and preferably 22 °C
  • the storage stable composition of the invention releases less than 1 % per month of the therapeutic agent from the conjugate, and (B) exhibits less than 1 % denaturation per month of the polypeptide binding moiety; when the composition is stored at 4°C in the absence of direct sunlight.
  • Methods for determining percentage release of therapeutic agent are well known in the art, for example HPLC (high-pressure liquid chromatography). Preferred method(s) are described in the present Examples section (below). Methods for determining percentage denaturation of the polypeptide binding moiety are also well known in the art, for example size exclusion chromatography, native gel electrophoresis or differential light scattering. Preferred method(s) are also described in the present Examples section (below).
  • polypeptide binding moiety we include polypeptides and proteins capable of reversibly and/or irreversibly associating with a region or regions of another molecule or molecules by covalent and/or ionic interaction.
  • therapeutic agent we include any purified or isolated natural or chemically-synthesised entity comprising one or more molecule which is pharmacologically active and exhibits a therapeutic effect when administered to a mammal, including human subjects.
  • the term includes one more polypeptide and/or one or more small chemical molecule, wherein said polypeptide and/or small chemical molecule may or may not be modified by the ionic and/or hydrophobic and/or covalent addition of chemical groups.
  • Therapeutic agents include but are not limited to low molecular weight drugs, nucleic acids, proteins, peptides and antigens.
  • Nucleic acids include, but are not limited to, DNA, cDNA, RNA, siRNA, RNAi.
  • Other large molecular weight therapeutic agents may include conjugates of such molecules, e.g., with polymers or cell-penetrating peptides.
  • the composition of the invention has a pH below 7.0 (for example 6.5 or below, 6.25 or below, 6.0 or below, 5.75 or below, 5.5 or below, 5.25 or below, 5.0 or below, or 4.75 or below).
  • the composition has a pH of 4.5 or above 4.5, for example at least pH 5.0, at least pH 5.25, at least pH 5.5, at least 5.75, at least 6.0 or at least pH 6.25.
  • the composition may have an acidic pH between 4.5 and 7.0, for example between 4.5 and 7.0, between 5.0 and 7.0, between 5 and 6.5, between 5 and 6.0, between 5.5 and 7.0, between 6.0 and 7.0, between or between 6.5 and 7.0.
  • the composition pH is between a lower pH and an upper pH, wherein the lower pH is selected from the group consisting of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3 and 6.4; and the upper pH is selected from the group consisting of 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3, 6.4 and 6.5; with the proviso that the upper pH is of a lower numerical value than the upper pH.
  • the composition pH is between 4.5 and 6.5, for example 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3, 6.4, 6.5.
  • the release rate of the therapeutic agent from the conjugate is less than 5% per month, for example less than 4%, 3%, 2%, 1%, 0.5% or less than 0.1% per month.
  • the polypeptide binding moiety denaturation rate of the conjugate is less than 5% per month, for example less than 4%, 3%, 2%, 1%, 0.5% or less than 0.1% per month.
  • the composition is stored at room temperature, for example between 20°C and 25°C. In another embodiment the composition is stored at between 0°C and 5°C, for example at 4°C (i.e., a chilled temperature). In a still further embodiment, the composition is store at above room temperature, for example, between 30°C and 37°C.
  • polypeptide binding moiety-therapeutic agent conjugate is represented by one or the following formulae:
  • R represents the therapeutic agent
  • R' represents the polypeptide binding moiety
  • the ester bond may be a dicarboxylic acid ester.
  • the dicarboxylic acid may comprise between 2 and 10 carbon atoms, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • the dicarboxylic acid is butanedioic acid.
  • the linker of the conjugate of the invention is absent. However, in another embodiment the linker of the conjugate of the invention is present.
  • the linker moiety serves to add flexibility to the protein-agent conjugates of the present invention, as well as ensuring appropriate separation between the two moieties.
  • the linker may be any molecular entity capable of joining two or more other molecules.
  • the linker of the conjugate of the invention may thus in one embodiment be a polymer, such as a hydrocarbon, for example a hydrocarbon selected from the group of hydrocarbons consisting of alkanes, alkenes and alkynes.
  • the linker is a polymer selected from the group of different types of polymers consisting of hydrocarbons, polyamides, polypeptides, polysacchrarides and polynucleotides.
  • the linker of the present invention may further comprise a tag for purification, such as the GST-tag or the hexahistidine tag (His6, 6H).
  • the linker comprises or consists of a peptide.
  • the linker is polypeptide.
  • the linker may thus be a polypeptide of any length suitable of performing the action of joining the first and the second part of the protein-agent conjugates of the present invention.
  • the linker polypeptide may be a peptide comprising at least 2 consecutive amino acid residues, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 at least 50 consecutive amino acid residues.
  • the peptide linker may be linear or circular, branched or unbranched. However, it is preferred that the peptide linker is linear and unbranched.
  • the polypeptide binding moiety is not linked to the ester bond via a glycine side chain in a very short peptide (2-5 residues).
  • the peptide linker comprises 6 or more consecutive amino acids.
  • linker peptide of the present invention may comprise at least 2-5 consecutive amino acid residues, at least 6-10 consecutive amino acid residues, such as at least 11- 15 consecutive amino acid residues, for example at least 16-20 consecutive amino acid residues, such as at least 21-25 consecutive amino acid residues, at least 26-30 consecutive amino acid residues, for example at least 31-35 consecutive amino acid residues, such as at least 36-40 consecutive amino acid residues, at least 41-45 consecutive amino acid residues, such as at least 46-50 consecutive amino acid residues.
  • the linker polypeptide may comprise at least 55 consecutive amino acid residues, at least 60 consecutive amino acid residues, such as at least 65 consecutive amino acid residues, for example at least 70 consecutive amino acid residues, such as at least 75 consecutive amino acid residues, at least 80 consecutive amino acid residues, for example at least 85 consecutive amino acid residues, such as at least 90 consecutive amino acid residues, for example at least 100 consecutive amino acid residues.
  • the linker peptide of the present invention comprise amino acids with properties suitable for being a flexible linker, while at the same time comprising a mixture of hydrophobic and hydrophilic amino acids, in a way that the linker will be soluble under different hydrophilic and hydrophobic conditions.
  • the linker peptide of the present invention may comprise any hydrophobic and any hydrophilic amino acid, and it is within the scope of the present invention that the linker comprises hydrophilic and hydrophobic amino acid residues in a ratio of at least 1 :1 , such as at least 1 :1.1 , for example at least 1 :1.2, at least 1 :1.3, at least 1 :1.4, at least 1 :1.5 for example at least 1 :1.6, at least 1 :1.7, at least 1 :1.8, at least 1 :1.9, for example at least 1 :2, at least 1 :2.1 , at least 1 :2.2, at least 1 :2.3, for example at least 1 :2.4, at least 1 :2.5, at least 1 :2.6, at least 1 :2.7, for example at least 1 :2.8, at least 1 :2.9, at least 1 :3, at least 1 :3.1 , at least 1 :3.2, at least 1 :3.3, at least 1 :3.4, at least 1 :3.5, at least 1
  • the linker comprises the amino acids serine (Ser, S) and glycine (Gly, G).
  • the mixture of these two amino acids will provide both flexibility, because of relative small size of these amino acids, and an optimal hydrophilicity owing to the nature of the hydroxylic side chain of serine.
  • the linker may in one embodiment comprise at least 20% glycine residues, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% glycine residues.
  • the linker may comprise at least 20% serine residues, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% serine residues.
  • the linker comprises or consists of alternating serine (Ser, S) and glycine (Gly, G) amino acids.
  • the linker sequence comprises or consists of the amino acid sequence 'SGSG' (i.e., 'Ser-Gly-Ser-Gly').
  • the polypeptide binding moiety is linked to the ester bond via a primary amino group of an amino acid within the polypeptide binding moiety.
  • the primary amino group may be on a side chain of an amino acid and it is preferred that the amino acid is lysine.
  • the primary amino group may be on a side chain of any amino acid within the polypeptide binding moiety.
  • the amino acid is not at the C- or N- terminus of the polypeptide binding moiety.
  • the polypeptide binding moiety is not linked to the ester bond via a primary amino group of a glycine side chain.
  • R-0 - represents the therapeutic agent
  • - N(H)-R' represents the polypeptide binding moiety.
  • the ester bond is degradable by an esterase (i.e., an enzyme that hydrolyses esters into an acid and an alcohol).
  • the esterase is a lysosomal esterase and/or an endosomal esterase.
  • lysosomal esterase we mean that the esterase is expressed in cells at a higher level within corresponding lysosomes than outside of the lysosomes.
  • endosomal esterase we mean that the esterase is expressed at a higher level within endosomes than outside of endosomes.
  • polypeptide binding moiety is a recombinant polypeptide.
  • polypeptide binding moiety is selected from the group consisting of:
  • antibody includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or constant regions, or other immunointeractive molecules capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.
  • antibody-like binding agents such as affibodies and aptamers.
  • one or more of the first binding molecules may be an aptamer (see Collett et a/., 2005, Methods 37:4-15).
  • Molecular libraries such as antibody libraries (Clackson et al, 1991 , Nature 352, 624-628; Marks et al, 1991 , J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985, Science 228(4705): 1315-7), expressed cDNA libraries (Santi et al (2000) J Mol Biol 296(2): 497- 508), libraries on other scaffolds than the antibody framework such as affibodies (Gunneriusson et al, 1999, Appl Environ Microbiol 65(9): 4134-40) or libraries based on aptamers (Kenan et al, 1999, Methods Mol Biol 118, 217-31) may be used as a source from which binding molecules that are specific for a given motif are selected for use in the methods of the invention.
  • the molecular libraries may be expressed in vivo in prokaryotic cells (Clackson et al, 1991 , op. tit; Marks et al, 1991 , op. cit.) or eukaryotic cells (Kieke et al, 1999, Proc Natl Acad Sci USA, 96(10): 5651-6) or may be expressed in vitro without involvement of cells (Hanes & Pluckthun, 1997, Proc Natl Acad Sci USA 94(10):4937-42; He & Taussig,
  • variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81 , 6851-6855).
  • variable domains that antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better er al (1988) Science 240, 1041); Fv molecules (Skerra er al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V H and V L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston er al (1988) Proc. Natl. Acad. Sci.
  • the antibody or antigen-binding fragment may be selected from the group consisting of intact antibodies, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments), single variable domains (e.g. V H and V L domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • the antibody or antigen-binding fragment is a single chain Fv (scFv).
  • scFv molecules we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • the antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference.
  • selector peptides having defined motifs are usually employed.
  • Amino acid residues that provide structure, decreasing flexibility in the peptide or charged, polar or hydrophobic side chains allowing interaction with the binding molecule may be used in the design of motifs for selector peptides. For example:
  • Proline may stabilise a peptide structure as its side chain is bound both to the alpha carbon as well as the nitrogen;
  • Phenylalanine, tyrosine and tryptophan have aromatic side chains and are highly hydrophobic, whereas leucine and isoleucine have aliphatic side chains and are also hydrophobic;
  • Lysine, arginine and histidine have basic side chains and will be positively charged at neutral pH, whereas aspartate and glutamate have acidic side chains and will be negatively charged at neutral pH;
  • Asparagine and glutamine are neutral at neutral pH but contain a amide group which may participate in hydrogen bonds;
  • Serine, threonine and tyrosine side chains contain hydroxyl groups, which may participate in hydrogen bonds.
  • selection of binding molecules may involve the use of array technologies and systems to analyse binding to spots corresponding to types of binding molecules.
  • the polypeptide binding moiety may comprise a detectable moiety.
  • the detectable moiety may be selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety and an enzymatic moiety.
  • the polypeptide binding moiety is an antibody or antigen-binding fragment thereof, or a variant, fusion or derivative thereof which retains the ability to bind antigen.
  • polypeptide binding moiety is an intact antibody, for example a monoclonal antibody.
  • the binding moiety may be a variant, fusion or derivative thereof of an antibody or antigen-binding fragment, provided such variants, fusions and derivatives retain binding specificity for a specific receptor, for example, CD163.
  • Variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001 , Cold Spring Harbor Laboratory Press, which is incorporated herein by reference).
  • 'fusion' we include an antibody or antigen-binding fragment (as defined herein) fused to any other polypeptide.
  • the antibody or antigen-binding fragment may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate its purification. Examples of such fusions are well known to those skilled in the art.
  • the said antibody or antigen-binding fragment may be fused to an oligo- histidine tag such as His6 or to an epitope recognised by a further antibody (such as the well-known Myc tag epitope).
  • the fusion may comprise a further portion which confers a desirable feature on the antibody or antigen-binding fragment of the invention; for example, the portion may be useful in detecting or isolating the antibody or antigen-binding fragment, or promoting cellular uptake of the antibody or antigen-binding fragment.
  • the portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art.
  • the moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake, as known to those skilled in the art.
  • the polypeptide binding moiety is an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv, disulphide-bonded Fv and domain antibodies), and Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments).
  • Fv fragments e.g. single chain Fv, disulphide-bonded Fv and domain antibodies
  • Fab-like fragments e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments.
  • the polypeptide binding moiety is an scFv.
  • the polypeptide binding moiety may be a domain antibody, such as a nanobody.
  • domain antibody we mean polypeptides corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies. Domain antibodies have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody. In contrast to conventional antibodies, domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. In addition, many dAbs are highly stable and retain activity even after being subjected to harsh conditions, such as freeze- drying or heat denaturation. These features make domain antibodies amenable to a wide range of pharmaceutical formulation conditions and manufacture processes. In addition, the small size of dAbs allows for higher molar quantities per gram of product, which can provide a significant increase in potency per dose and reduction in overall manufacturing cost.
  • the domain antibodies selected against a particular target can be used as a building block to create therapeutic products with unique characteristics not available to conventional antibodies or proteins, such as dual-targeting dAbs that bind to two therapeutic targets in one easily produced molecule, dAbs with a tailored serum half life, dAbs for pulmonary or oral administration for lung or Gl tract diseases, and dAbs against targets that can not be easily addressed by IgGs.
  • Such antibodies can be produced recombinantly but are also are known to exist in camelids (Curr. Opin. Pharmacol., 8, (2008), 600-608) and sharks (e.g. IgNAR; Curr. Opin. Pharmacol., 8, (2008), 600-608).
  • Other preferred antibody variants include isolated heavy-chain variable (V H ) regions or isolated light-chain (V L ) regions, for example from human antibodies (Curr. Opin. Pharmacol., 8, (2008), 600-608), and iMabs (WO 03/050283).
  • the domain antibody may be selected from the group consisting of single-domain antibodies from cameloids, single domain antibodies from sharks and isolated V H or V L domains from humans.
  • antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991 , Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture.
  • these include, but are not limited to, the hybridoma technique, the human B- cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler et al., 1975.
  • the domain antibody is selected from the group consisting of single domain antibodies from cameloids, single domain antibodies from sharks and isolated V H or V L domains from humans.
  • the antibody may be an IgG antibody, for example, an lgG2 or lgG4 antibody. It is preferred that the antibody is human or humanised.
  • humanised antibodies may be used for human therapy or diagnostics.
  • Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimaeric antibodies or antibody fragments having minimal-portions derived from non-human antibodies.
  • Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non human species (donor antibody) such as mouse, rat of rabbit having the desired functionality.
  • donor antibody such as mouse, rat of rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported Complementarity Determining Region (CDR) or framework sequences.
  • CDR Complementarity Determining Region
  • the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et a/., 1986. Nature 321 :522-525; Riechmann et a/., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596, which are incorporated herein by reference).
  • the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et a/., 1986, Nature 321 :522-525; Reichmann ei a/., 1988. Nature 332:323-327; Verhoeyen et a/., 1988, Science 239:1534-15361; US 4,816,567, which are incorporated herein by reference) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions.
  • humanised antibodies are chimaeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991 , J. Mol. Biol. 227:381 ; Marks er a/., 1991 , J. Mol. Biol. 222:581 ; Cole er a/., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner er a/., 1991. J. Immunol. 147:86-95, Soderlind et a/., 2000, Nat Biotechnol 18:852-6 and WO 98/32845 which are incorporated herein by reference).
  • phage display libraries see, for example, Hoogenboom & Winter, 1991 , J. Mol. Biol. 227:381 ; Marks er a/., 1991 , J. Mol. Biol. 222:581 ; Cole er a/., 1985, In: Monoclo
  • suitable antibodies may be tested for activity, such as binding specificity or a biological activity of the antibody, for example by ELISA, immunohistochemistry, flow cytometry, immunoprecipitation, Western blots, ere.
  • activity such as binding specificity or a biological activity of the antibody, for example by ELISA, immunohistochemistry, flow cytometry, immunoprecipitation, Western blots, ere.
  • the biological activity may be tested in different assays with readouts for that particular feature.
  • the polypeptide binding domain is an antibody mimic.
  • antibody mimics for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions
  • antibody mimics may be used to create molecular libraries from which binding moieties can be derived.
  • binding moieties may be derived from many skilled in the arts of biochemistry.
  • Such molecules may be used as a binding moiety in the agent of the present invention.
  • Suitable antibody mimics and methods for their production are discussed in Skerra et al. (2007, Curr. Opin. Biotech., 18: 295-304), Nuttall & Walsh (2008, Curr. Opin. Biotech., 8: 309-615), and Gill & DAmle (2006, Curr. Opin. Biotech., 17: 653-8).
  • Exemplary antibody mimics include: affibodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol.
  • the antibody mimic is selected from the group comprising or consisting of affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
  • the binding moiety is an RNA aptamer.
  • RNA aptamers represent a unique emerging class of therapeutic agents (Que-Gewirth et al, Gene Ther. 74:283 (2007); Ireson et al, Mol. Cancer Ther. 5:2957 (2006)). They are relatively short (12-30 nucleotide) single-stranded RNA oligonucleotides that assume a stable three-dimensional shape to tightly and specifically bind selected protein targets to elicit a biological response. In contrast to antisense oligonucleotides, RNA aptamers can effectively target extracellular targets. Like antibodies, aptamers possess binding affinities in the low nanomolar to picomolar range.
  • aptamers are heat stable, non-immunogenic, and possess minimal inter-batch variability. Chemical modifications, such as amino or fluoro substitutions at the 2' position of pyrimidines, may reduce degradation by nucleases. The biodistribution and clearance of aptamers can also be altered by chemical addition of moieties such as polyethylene glycol and cholesterol. Aptamers may be developed by iterative selection methods such as SELEX (systematic evolution of ligands by exponential enrichment) to specifically recognize and tightly bind their targets by means of well-defined complementary three-dimensional structures. Further, SELEX (and other such methods) allows selection from libraries to generate high-affinity oligonucleotide ligands to purified biochemical targets.
  • SELEX systematic evolution of ligands by exponential enrichment
  • RNA aptamers have been demonstrated to target the Ku DNA repair proteins with resulting sensitization of breast cancer cells to etoposide (Zhang et al, Int. J. Mol. Med. 74:153 (2004)).
  • the binding moiety is not an RNA aptamer.
  • the binding moiety is a haptoglobin-hemoglobin (Hp-Hb) complex.
  • CD163 mediates the clearance of the Hp-Hb complexes formed when hemoglobin is librated to the circulation during intravascular hemolysis and it is also involved in regulation of inflammatory processes.
  • CD163 is considered to be expressed exclusively on the surface of the monocytic lineage. It is expressed by resident monocytes in the circulation and upregulated during maturation to macrophages and is highly expressed on tissue-resident macrophages, as well as on alternatively activated macrophages (M2), and TIE2+ macrophages.
  • M2 alternatively activated macrophages
  • TIE2+ macrophages TIE2+ macrophages
  • Hp-Hb complexes The production and use of Hp-Hb complexes is described in WO 02/32941.
  • the antibody of the therapeutic agents of the invention may bind to any tissue- or cell-specific antigen or epitope. It is preferred that the antibody binds to the antigen or epitope selectively.
  • the antigen or epitope may be associated with a disease or condition.
  • the disease or condition is selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • the disease or condition is an inflammatory disease selected from the group consisting of: arthritic diseases (such as rheumatoid arthritis, spondylitis, osteoarthritis); chronic inflammatory bowel disease (IBD, such as Crohn's disease, ulcerative colitis); peridontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue disease; autoimmune liver disease (such as biliary cirrhosis); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes; obesity; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; joint inflammation; inflammation-induced cartilage destruction; liver cirrhosis; organ transplantation; Idiopathic Thrombocytopenic Purpura (ITP);
  • the antibody, antigen-binding fragment, variant, fusion or derivative thereof is derived from an antibody selected from Mac2-158, Mac2-48 and Exemplary humanised mAb.
  • epitope it is herein intended to mean a site of a molecule to which an antibody binds, i.e. a molecular region of an antigen.
  • An epitope may be a linear epitope, which is determined by e.g. the amino acid sequence, i.e. the primary structure, or a three- dimensional epitope, defined by the secondary structure, e.g. folding of a peptide chain into beta sheet or alpha helical, or by the tertiary structure, e.g. way which helices or sheets are folded or arranged to give a three-dimensional structure, of an antigen.
  • CD163 is a membrane receptor molecule expressed on macrophages, which functions as an endocytic receptor for haemoglobin-haptoglobin-complexes. By this physiologically very important function, CD163 each day takes up approximately 1g of haemoglobin, and the protein therefore is probably the most highly expressed receptor on macrophages.
  • Macrophages are part of the innate immune defence and play a central role in many infectious, autoimmune, and malignant diseases.
  • autoimmune/inflammatory disease such as Rheumatoid Arthritis
  • macrophages are the main source of inflammatory molecules such as TNF-alpha, known to be of crucial importance in disease progression.
  • infectious diseases such as TB and HIV
  • macrophages harbour the infectious agent.
  • a few malignant diseases have their origin in cells of the monocytic/macrophage lineage such as histiocytic sarcoma.
  • the polypeptide binding moiety is capable of binding selectively to a protein expressed on the surface of a cell.
  • binding moiety may be capable of binding selectively to a specific protein type under physiological conditions, e.g. in vivo. Suitable methods for measuring relative binding strengths include immunoassays, for example where the binding moiety is an antibody (see Harlow & Lane, “Antibodies: A Laboratory”, Cold Spring Habor Laboratory Press, New York, which is incorporated herein by reference). Alternatively, binding may be assessed using competitive assays or using Biacore ® analysis (Biacore International AB, Sweden).
  • the cell is selected from the group consisting of monocytes, macrophages, monocyte-derived dendritic cells, activated macrophage subtypes (for example M1 and M2) and malignant cells.
  • the cell is a monocyte or monocyte-derived cell.
  • the cell may be a macrophage, such as a Kuppfer cell.
  • the cell expresses CD163 at its surface.
  • Direct targeting of drugs to macrophages can have a significant impact on certain diseases without influencing other cells in the body.
  • the macrophage targeting of drugs to act in the macrophage may therefore reduce side-effects caused by effects on other cell types. Targeting can therefore increase the therapeutic index of the drug.
  • monocytes are mononuclear phagocytic cells that act within the immune system - that is, they are white blood cells having a single nucleus, which are capable of ingesting foreign material. Monocytes migrate from blood into tissues of the body and differentiate into cells such as macrophages. Thus, by "monocyte-derived cell", we include those cell types that have differentiated from monocytes.
  • the polypeptide binding moiety is capable of binding selectively to a receptor.
  • a receptor By “capable of binding selectively to a receptor” we include such antibody-derived binding moieties which bind at least 10-fold more strongly to a specific receptor type to another protein type; for example at least 50-fold more strongly or at least 100-fold more strongly.
  • the binding moiety may be capable of binding selectively to the specific receptor type under physiological conditions, e.g. in vivo.
  • receptor we include any biological molecule that serves to bind to a specific ligand or ligands under physiological conditions.
  • the receptor is a cell surface receptor.
  • CovX-Bodies are created by covalently joining a pharmacophore via a linker to the binding site of a specially-designed antibody, effectively reprogramming the antibody (Tryder et a/., 2007, Bioorg. Med. Chem. Lett, 17:501-6).
  • the result is a new class of chemical entities that is formed where each component contributes desirable traits to the intact CovX-Body - in particular, the entity has the biologic actions of the peptide and the extended half-life of the antibody.
  • the receptor is selected from the group consisting of: CD163; a mannose receptor; MARCO; M160; a complement receptor specific for the macrophage (such as an Fc receptor); a Siglec receptor, such as Siglec 1 , Siglec 5 and Siglec 1 1.
  • the receptor is CD163, particularly human CD163.
  • the CD163 is selected from the group of proteins defined by database accession nos. CAB45233, AAY99762, AAH51281 , EAW8862, EAW8863, EAW8864, EAW8865, EAW8866, NP_004235, NP_98161 and Swiss-Prot: Q86VB7.1.
  • Database accession no. AAH51281. NP 004325 and Swiss-Prot. Q86VB7.1 Database accession no. AAH51281. NP 004325 and Swiss-Prot. Q86VB7.1 :
  • the CD163 is localised on the surface of a cell.
  • CD163 is a scavenging receptor consisting of nine extracellular scavenger receptor cysteine-rich (SRCR) type B domains. It mediates the clearance of the haptoglobin-hemoglobin (Hp-Hb) complexes formed when hemoglobin is librated to the circulation during intravascular hemolysis is involved in regulation of inflammatory processes.
  • SRCR scavenger receptor cysteine-rich
  • CD163 is considered to be expressed exclusively on the surface of the monocytic lineage. It is expressed by resident monocytes in the circulation and upregulated during maturation to macrophages.
  • the polypeptide binding moiety is an antibody or antigen-binding fragment thereof with binding specificity for CD163, or a variant, fusion or derivative of said antibody or an antigen-binding fragment, or a fusion of a said variant or derivative thereof, or a fusion of a said variant or derivative thereof, which retains the binding specificity for CD163.
  • suitable methods for measuring relative binding strengths include immunoassays, for example where the binding moiety is an antibody (see Harlow & Lane, “Antibodies: A Laboratory”, Cold Spring Habor Laboratory Press, New York, which is incorporated herein by reference).
  • binding may be assessed using competitive assays or using surface plasmon resonance analysis such as Biacore ® analysis (Biacore International AB, Sweden).
  • the antibody or antigen-binding fragment, or variant, fusion or derivative thereof is capable of binding to CD163.
  • the variant, fusion or derivative of the antibody or antigen- binding fragment exhibits a binding affinity (Kd) of 50% or more of the binding affinity of the antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment, or variant, fusion or derivative thereof binds to a domain of CD163 selected from the group consisting of domain 1 , domain 2, domain 3, domain 4, domain 5, domain 6, domain 7, domain 8 and domain 9.
  • the antibody or antigen-binding fragment, or variant, fusion or derivative thereof binds to domain 1 of CD163.
  • the antibody or antigen-binding fragment, or variant, fusion or derivative thereof binds to domain 3 of CD163.
  • the antibody or antigen-binding fragment, or variant, fusion or derivative thereof exhibits greater binding affinity for CD163 in the presence of calcium than in the absence of calcium.
  • the term 'antibody' also includes all classes of antibodies, including IgG, IgA, IgM, IgD and IgE.
  • the antibody may be an IgG molecule, such as an lgG1 , lgG2, lgG3, or lgG4 molecule.
  • the antibody is an IgG antibody, for example, an lgG2 or lgG4 antibody.
  • the antibody is an lgG4 antibody in which the Serine amino acid at position 241 has been substituted with a Proline residue (i.e. S241 P) - such a substitution is known to stabilise the disulphide bridges in lgG4 molecule, resulting in a more stable antibody (Angal et al., 1993, Mol. Immunol., 30:105-8).
  • the antibody, antigen-binding fragment, variant, fusion or derivative thereof is in an isolated and/or purified form.
  • the antibody is a non-naturally occurring antibody.
  • the antibody is a naturally occurring antibody, it is provided in an isolated form (i.e. distinct from that in which it is found in nature).
  • the therapeutic agent is internalised into the cell when bound to CD163.
  • intercellular internalisation of molecules we include the molecular, biochemical and cellular events associated with the process of translocating a molecule from the extracellular surface of a cell to the intracellular surface of a cell.
  • the processes responsible for intracellular internalisation of molecules is well-known to those skilled in the field of molecular and cellular biology and can involve the internalisation of extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-associated molecules (such as cell-surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (for example, a ligand bound to a transmembrane receptor or an antibody bound to a membrane-associated molecule).
  • the therapeutic agent has efficacy in the treatment and/or prevention of a disease or condition selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • 'treatment' we include both therapeutic and prophylactic treatment of a subject/patient.
  • 'prophylactic' is used to encompass the use of an agent, medicament or pharmaceutical formulation described herein which either prevents or reduces the likelihood of a disease or condition in a patient or subject.
  • the appropriate dosage of an agent will depend on the type of condition or disorder to be treated, the severity and of course of the condition or disorder, whether the agent is administered for prophylactic or therapeutic purposes, the course of previous therapy and the patient's clinical history and response to the agent.
  • the effectiveness of an agent of the invention in alleviating the symptoms, preventing or treating a condition or disorder may be improved by serial administering or administration in combination with another agent that is effective for the same condition or disorder, such as conventional therapeutic agents known for the intended therapeutic indication.
  • the therapeutic agent has efficacy in the treatment and/or prevention of acute and/or chronic inflammatory diseases.
  • the therapeutic agent may have efficacy in the treatment and/or prevention of an autoimmune disease.
  • the immune disease or condition may be selected from the group consisting of: arthritic diseases (such as rheumatoid arthritis, spondylitis, osteoarthritis); chronic inflammatory bowel disease (IBD, such as Crohn's disease, ulcerative colitis); peridontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue diesase; autoimmune liver disease (such as biliary cirrhosis); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes; obesity; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; joint inflammation; inflammation-induced cartilage destruction; liver cirrhosis; organ transplantation; Idiopathic Thrombocytopenic Purpura (ITP);
  • the immune disease or condition is selected from the group consisting of sepsis, non-viral hepatitis, alcoholic steatohepatitis (ASH) or acute alcoholic hepatitis.
  • Glucocorticoids are the most effective anti-inflammatory drugs known, and are often used in the early phases of Rheumatoid Arthritis (RA) and for long- term use at low dose in a large fraction of patients. When used for long-term treatment, however, glucocorticoids have severe adverse side effects (e.g. osteoporosis, muscle atrophy, suppression of the adrenals, increased risk of infection, growth retardation, type 2 diabetes and more) that limits chronic treatment.
  • the therapeutic agent may be an immunosuppressive agent, such as an anti- inflammatory agent.
  • the immunosuppressive agent may be selected from the group comprising or consisting of: a glucocorticoid; corticosteroid; methotrexate; cyclophosphamide; 6-mercaptopurin; cyclosporine; tacrolimus; mycophenolate mofetil; sirulimus; everolimus; an siRNA molecule capable of inhibiting synthesis of proinflammatory cytokines (such as TNF); a non-steroidal anti-inflammatory therapeutic agent (NSAIDs, such as aspirin, ibuprofen); a steroid (such as vitamin D); a disease- modifying anti-rheumatic therapeutic agent (DMARDs, such as penicillamin, sulfasalazin, cyclosporine).
  • a glucocorticoid corticosteroid
  • methotrexate cyclophosphamide
  • the immunosuppressive agent is a glucocorticoid.
  • Exemplary glucocorticoids may be selected from the group comprising or consisting of: cortisone and derivatives thereof (such as hydrocortisone); prednisone and derivatives thereof (such as prednisolone, methylprednisolone, methylprednisolone-acetate, methylprednisolone-succinate); dexamethasone and derivatives thereof; triamcinolone and derivatives thereof (such as triamcinolonehexacetonuid, triamcinolonacetonamid); paramethasone; betamethasone; fluhydrocortisone; fluocinolone acetonide; fluocinolone.
  • cortisone and derivatives thereof such as hydrocortisone
  • prednisone and derivatives thereof such as prednisolone, methylprednisolone, methylprednisolone-acetate, methylprednisolone-succinate
  • dexamethasone and derivatives thereof
  • the glucocorticoid is prednisone. In a further embodiment, the glucocorticoid is methyl-prednisoline. In a still further embodiment, the glucocorticoid is fluocinolone-acetonoid.
  • the immunosuppressive agent is preferably dexamethasone. In another embodiment, the immunosuppressive agent is a glucocorticoid-hemisuccinate derivative.
  • agents of the compositions of the invention may further comprise a detectable and/or cytotoxic moiety.
  • the therapeutic agent has efficacy in the treatment and/or prevention of infection.
  • the infection may be selected from the group consisting of: Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Venezuelan hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (Noro)
  • the infection is Human Immunodeficiency Virus (HIV). It is also preferred that the infection is tuberculosis.
  • the tuberculosis is caused by infection with Mycobacterium tuberculosis.
  • the tuberculosis may alternatively or additionally be caused by infection with one or more of Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti or Mycobacterium microti.
  • the therapeutic agent may be an antimicrobial agent, such as an antibacterial, an antimycotic, an antimycobacterial or an antiviral.
  • the therapeutic agent is an antibacterial.
  • the antibacterial may be selected from the group consisting of Demeclocycline, Doxycycline, Chlortetracycline, Lymecycline, Metacycline, Oxytetracycline, Tetracycline, Minocycline, Rolitetracycline, Penimepicycline, Clomocycline, Tigecycline, Chloramphenicol, Thiamphenicol, Florfenicol, Ampicillin, Pivampicillin, Carbenicillin, Amoxicillin, Carindacillin, Bacampicillin, Epicillin, Pivmecillinam, Azlocillin, Mezlocillin, Mecillinam, Piperacillin, Ticarcillin, Metampicillin, Talampicillin, Sulbenicillin, Temocillin, Hetacillin, Benzylpenicillin, Phenoxymethylpenicillin, Propicillin, Azidocillin, Pheneticillin, Penamecillin, Clometocillin, Benzathine benz
  • the therapeutic agent is an antimycotic.
  • the antimycotic may be selected from the group consisting of Amphotericin B, Hachimycin, Miconazole, Ketoconazole, Clotrimazole, Fluconazole, Itraconazole, Voriconazole, Posaconazole, Flucytosine, Caspofungin, Micafungin and Anidulafungin.
  • the therapeutic agent is an antimycobacterial.
  • the antimycobacterial may be selected from the group consisting of Aminosalicylic acid, Sodium aminosalicylate, Calcium aminosalicylate, Cycloserine, Rifampicin, Rifamycin, Rifabutin, Rifapentin, Capreomycin, Isoniazid, Protionamide, Tiocarlide, Ethionamide, Pyrazinamide, Ethambutol, Terizidone, Morinamide, Dapsone and Aldesulfone sodium.
  • the therapeutic agent is an antiviral.
  • the antiviral may be selected from the group consisting of Metisazone, Aciciovir, Idoxuridine, Vidarabine, Ribavirin, Ganciclovir, Famciclovir, Valaciclovir, Cidofovir, Penciclovir, Valganciclovir, Brivudine, Rimantadine, Tromantadine, Foscarnet, Fosfonet, Saquinavir, Indinavir, Ritonavir, Nelfinavir, Amprenavir, Lopinavir, Fosamprenavir, Atazanavir, Tipranavir, Darunavir, Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Tenofovir disoproxil, Adefovir dipivoxil, Emtricitabine, Entecavir, Telbivudine, Clevudin
  • detectable moiety we include the meaning that the moiety is one which, when located at the target site following administration of an agent of the invention to a patient, may be detected, typically non-invasively from outside the body and the site of the target located.
  • the detectable moiety may be a single atom or molecule which is either directly or indirectly involved in the production of a detectable species.
  • the agents of this embodiment of the invention are useful in imaging and diagnosis.
  • detectable moieties are well known in medicinal chemistry and the linking of these moieties to polypeptides and proteins is well known in the art.
  • detectable moieties include, but are not limited to, the following: radioisotopes (e.g. 3 H, 4C, 35 S, 123 l, 125 l, 13 l, "Tc, 111 ln, 90 Y, 188 Re), radionuclides (e.g. 11 C, 18 F, 6 Cu), fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphors, carbocyanine), enzymatic labels (e.g.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • the detectable moiety comprises a radioactive atom, such as a radioactive atom selected from the group consisting of: technetium-99; technitium-99m; iodine-123; iodine-124; iodine-131 ; indium-1 1 1 ; fluorine-18; fluorine-19; carbon-11 ; carbon-13; copper-64; nitrogen-13; nitrogen-15; oxygen-15; oxygen-17; arsenic-72; gadolinium; manganese; iron; deuterium; tritium; yttrium-86; zirconium-89.
  • a radioactive atom selected from the group consisting of: technetium-99; technitium-99m; iodine-123; iodine-124; iodine-131 ; indium-1 1 1 ; fluorine-18; fluorine-19; carbon-11 ; carbon-13; copper-64; nitrogen-13; nitrogen-15; oxygen-15; oxygen-17; arsenic-72; gadolin
  • the radio- or other labels may be incorporated into the agents of the invention in known ways.
  • the binding moiety is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as 99m Tc, 123 l, 186 Rh, 188 Rh and 111 ln can, for example, be attached via cysteine residues in the binding moiety.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm.
  • the invention provides an agent further comprising a cytotoxic moiety.
  • cytotoxic moiety we include the meaning that the moiety is one which is capable of inducing cell death in vivo or in vitro, for example when administered to a patient.
  • the cytotoxic moiety may be a single atom or molecule which is either directly or indirectly involved in inducing cell death.
  • the agents of this embodiment of the invention are useful in therapy (for example, where it is desired to remove or destroy one or more cell in an individual).
  • Suitable cytotoxic moieties are well known in medicinal chemistry and the linking of these moieties to polypeptides and proteins is well known in the art.
  • the two portions may be linked together by any of the conventional ways of cross-linking polypeptides, such as those generally described in O'Sullivan et al (1979) Anal. Biochem. 100, 100-108.
  • the binding moiety may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP).
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • a bifunctional agent capable of reacting with those thiol groups
  • cytotoxic moieties include, but are not limited to, the following: radioisotopes (e.g. 123 l, 125 l, 3 l, 1 1 ln, 90 Y), alkylating agents (e.g. cisplatin), antimetabolites (e.g. methotrexate), antimitotics (e.g. vincristine), topoisomerase inhibitors (e.g. etoposide), and toxins (e.g. calicheamicin).
  • radioisotopes e.g. 123 l, 125 l, 3 l, 1 1 ln, 90 Y
  • alkylating agents e.g. cisplatin
  • antimetabolites e.g. methotrexate
  • antimitotics e.g. vincristine
  • topoisomerase inhibitors e.g. etoposide
  • toxins e.g. calicheamicin
  • the cytotoxic moiety comprises a radioactive atom, such as a radioactive atom selected from the group consisting of: iodine-123; iodine-125; iodine- 131 ; indium-111; bromine-77; copper-67; arsenic-77; astatine-211 ; actinium-225; bismuth-212; bismuth-213; lutetium-177; holmium-166; phosphorous-33; platinum-193; platinum-195; rhenium-186; rhenium-188; strontium-89; yttrium-90.
  • a radioactive atom selected from the group consisting of: iodine-123; iodine-125; iodine- 131 ; indium-111; bromine-77; copper-67; arsenic-77; astatine-211 ; actinium-225; bismuth-212; bismuth-213; lutetium-177; holm
  • the cytotoxic moiety comprises a drug selected from the group consisting of: an alkylating agent (such as cisplatin, carboplatin); an antimetabolite (such as azathioprine, methotrexate); an antimitotic drug (such as vincristine); a topoisomerase inhibitor (such as doxorubicine, etoposide); a toxin (such as calicheamicin).
  • an alkylating agent such as cisplatin, carboplatin
  • an antimetabolite such as azathioprine, methotrexate
  • an antimitotic drug such as vincristine
  • a topoisomerase inhibitor such as doxorubicine, etoposide
  • a toxin such as calicheamicin
  • composition according to any one of the preceding claims further comprising a pharmaceutically-acceptable diluent, carrier or excipient.
  • the composition may be a pharmaceutical composition.
  • composition means a therapeutically effective formulation according to the invention.
  • a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
  • This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host.
  • the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount of the active component is provided.
  • a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • the agents, medicaments and pharmaceutical compositions of the invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
  • An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • delivery is performed intra-muscularly (i.m.) and/or sub-cutaneously (s.c.) and/or intravenously (i.v.).
  • the agents, medicaments and pharmaceutical compositions of the invention can be administered by a surgically implanted device that releases the drug directly to the required site.
  • a surgically implanted device that releases the drug directly to the required site.
  • Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis.
  • the direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
  • Electroporation therapy (EPT) systems can also be employed for the administration of the agents, medicaments and pharmaceutical compositions of the invention.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • the agents, medicaments and pharmaceutical compositions of the invention can also be delivered by electro-incorporation (El).
  • El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin.
  • the particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • ReGel injectable system that is thermo-sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active substance is delivered over time as the biopolymers dissolve.
  • the agents, medicaments and pharmaceutical compositions of the invention can also be delivered orally.
  • the process employs a natural process for oral uptake of vitamin Bi 2 and/or vitamin D in the body to co-deliver proteins and peptides.
  • the agents, medicaments and pharmaceutical compositions of the invention can move through the intestinal wall.
  • Complexes are synthesised between vitamin B 12 analogues and/or vitamin D analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B 2 portion/vitamin D portion of the complex and significant bioactivity of the active substance of the complex.
  • the agents, medicaments and pharmaceutical compositions of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al. (1998), Trends Cell Biol 8, 84-87.
  • the medicaments and/or pharmaceutical compositions of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
  • agents, medicaments and pharmaceutical compositions of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • the compositions may be administered at varying doses.
  • the agents, medicaments and pharmaceutical compositions of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the agents, medicaments and pharmaceutical compositions of the invention can be administered orally, buccally or sublingually in the form of capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the agents, medicaments and pharmaceutical compositions of the invention may also be administered via intracavernosal injection.
  • the liquid compositions of the invention may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the agents, medicaments and pharmaceutical compositions of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the agents, medicaments and pharmaceutical compositions of the invention can be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the medicaments and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials.
  • the daily dosage level of the agents, medicaments and pharmaceutical compositions of the invention will usually be from 5 to 1500 mg per adult per day administered in single or divided doses.
  • the agents, medicaments and pharmaceutical compositions of the invention may contain from 5mg to 1400mg (for example, from 7mg to 1400mg, or 5mg to 1000mg) and may preferably contain 5mg to 200mg of active agent for administration singly or two or more at a time, as appropriate.
  • the agents, medicaments and pharmaceutical compositions of the invention are administered at a dosage ranging from 0.02mg/kg to 2mg/kg and at a frequency ranging from twice per week to once per month.
  • agents, medicaments and pharmaceutical compositions of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of an inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA 134A3 or 1 ,1 , 1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of an agent of the invention and a suitable powder base such as lactose or starch.
  • Aerosol or inhaled formulations are preferably arranged so that each metered dose or "puff contains at least 1 mg of an agent of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the agents, medicaments and pharmaceutical compositions of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, gel or ointment.
  • the agents, medicaments and pharmaceutical compositions of the invention may also be transdermal ⁇ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.
  • the agents, medicaments and pharmaceutical compositions of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • the agents, medicaments and pharmaceutical compositions of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water.
  • ком ⁇ онентs can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia
  • mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • oral or parenteral administration of the agents, medicaments and pharmaceutical compositions of the invention is the preferred route, being the most convenient.
  • the agents, medicaments and pharmaceutical compositions of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • the agents of the invention may be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used, for example as described in the accompanying Examples.
  • formulations may comprise a lower concentration of a compound of the invention.
  • the present invention provides a pharmaceutical composition comprising an amount of a protein-agent conjugate of the invention effective to treat various conditions (as described above and further below).
  • the composition is for delivery parenterally (for example, intravenously, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously), intranasally, by inhalation or intraocularly.
  • the present invention also includes pharmaceutical compositions comprising pharmaceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention.
  • the acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e.
  • salts containing pharmacologically acceptable anions such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1 ,1'-methylene-bis-(2- hydroxy-3 naphthoate)] salts, among others.
  • pharmacologically acceptable anions such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate,
  • Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents according to the present invention.
  • the chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present agents that are acidic in nature are those that form non-toxic base salts with such compounds.
  • Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N- methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
  • the composition of the invention is for use in medicine.
  • the composition may be for use in the treatment or prevention of a disease or condition selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • a disease or condition selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • the composition is for use in the treatment or prevention of an acute and/or chronic inflammatory disease or condition.
  • the composition is for use in the treatment or prevention of an autoimmune disease.
  • the disease or condition is selected from the group consisting of: arthritic diseases (such as rheumatoid arthritis, spondylitis, osteoarthritis); chronic inflammatory bowel disease (IBD, such as Crohn's disease, ulcerative colitis); peridontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue disease; autoimmune liver disease (such as biliary cirrhosis); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes; obesity; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; joint inflammation; inflammation-induced cartilage destruction; liver cirrhosis; organ transplantation; Idiopathic Thrombocytopenic Purpura (ITP); sarco
  • the disease or condition is rheumatoid arthritis.
  • the composition of the invention is for use in the treatment or prevention of acute inflammation.
  • a second aspect of the invention provides the use of a composition according to any one of the preceding claims in the preparation of a medicament for the treatment or prevention of a disease or condition selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • the use is for the treatment or prevention of an acute and/or chronic inflammatory disease or condition.
  • the use is for the treatment or prevention of an autoimmune disease.
  • the immune disease or condition is selected from the group consisting of: arthritic diseases (such as rheumatoid arthritis, spondylitis, osteoarthritis); chronic inflammatory bowel disease (IBD, such as Crohn's disease, ulcerative colitis); peridontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue disease; autoimmune liver disease (such as biliary cirrhosis); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes; obesity; nonalcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; joint
  • the use is for the treatment or prevention of rheumatoid arthritis.
  • the use may equally be for the treatment or prevention of acute inflammation.
  • the disease or condition is infection.
  • the infection may be selected from the group consisting of: Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Venezuelan hemorrhagic fever,
  • the infection is Human Immunodeficiency Virus (HIV). It is also preferred that the infection is tuberculosis.
  • the tuberculosis is caused by infection with Mycobacterium tuberculosis.
  • the tuberculosis may alternatively or additionally be caused by infection with one or more of Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti or Mycobacterium microti.
  • a third aspect of the invention provides a method for the treatment or prevention of a disease or condition in a patient wherein the disease or condition selected from the group consisting of acute and chronic inflammatory diseases and conditions, and infectious diseases.
  • the disease or condition is an acute and/or chronic inflammatory disease or condition.
  • the disease or condition may be an autoimmune disease.
  • the disease or condition is selected from the group consisting of: arthritic diseases (such as rheumatoid arthritis, spondylitis, osteoarthritis); chronic inflammatory bowel disease (IBD, such as Crohn's disease, ulcerative colitis); peridontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue disease; autoimmune liver disease (such as biliary cirrhosis); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes; obesity; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; joint inflammation; inflammation-induced cartilage
  • the disease or condition is rheumatoid arthritis. Also preferably, the disease or condition is acute inflammation.
  • the disease or condition is infection.
  • the infection may be selected from the group consisting of: Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Venezuelan hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis.urkholderia infection, Buruli
  • a fourth aspect of the invention provides a method of making a composition of the invention comprising conjugating a polypeptide binding moiety to a therapeutic agent via an ester bond.
  • the polypeptide binding moiety is conjugated to a therapeutic agent indirectly via a linker comprising an ester bond.
  • the method may comprise the step of adjusting the pH of the composition, as necessary, to make it acidic.
  • Figure 1 shows how an ester linkage can connect a targeting agent with a drug or prodrug, including targeting (agent) and linker.
  • CO-0 is the ester and drug is the drug or prodrug.
  • Two ways of arranging the ester can be envisioned, A and B.
  • the biodegradable ester is marked with an arrow.
  • Figure 2 shows the synthesis of dexamethasone-hs-NHS from dexamethasone and the structure of dexamethasone hemisuccinate linked to an antibody through the amino group of a lysine.
  • Figure 3 shows the release profile of dexamethasone (% of total dexamethasone) from human IgG-dexamethasone conjugates after storage in buffers with different pH at 37°C.
  • Figure 4 shows the release profile of dexamethasone (% of total dexamethasone) from human IgG-dexamethasone conjugates after storage in buffers with different pH at 4°C.
  • Figure 5 shows the release profile of dexamethasone (%of total dexamethasone) from human IgG-dexamethasone conjugates after storage in buffers with different acidic pH at 4°C.
  • Figure 6 protein recovery (protein in solution at indicated time point in relation to time point 0) in % in hlgG-NHS-dexamethasone-conjugates stored in buffers with different pH at 37°C.
  • Figure 7 Fluocinolone-Acetonide (% from total) released from 3E10B10-fluocinolone- acetonide-conjugates stored in buffers with different pH at 4°C.
  • Figure 8 shows the release profile of dexamethasone (%of total dexamethasone) from KN2/NRY-dexamethasone conjugates after storage in buffers with different pH at 37°C.
  • Figure 9 shows the release profile of dexamethasone (%of total dexamethasone) from KN2/NRY-dexamethasone conjugates after storage in buffers with different pH at 4°C.
  • Figure 10 shows the release profile of dexamethasone (%of total dexamethasone) from KN2/NRY-dexamethasone conjugates after storage in buffers with different acidic pH at 4°C.
  • Figure 11 Analytical gelfiltration of KN2/NRY-NHS-dexamethasone-conjugate stored in buffer of pH 4.0 at 37°C.
  • Figure 12 Analytical gelfiltration of KN2/NRY-NHS-dexamethasone-conjugate stored in buffer of pH 5.0 at 37°C.
  • Figure 13 Analytical gelfiltration of KN2/NRY-NHS-dexamethasone-conjugate stored in buffer of pH 6.5 at 37°C.
  • Dexamethasone (1.00 g, 2.55 mmol) and succinic anhydride (1.27 g, 12.69 mmol) was stirred overnight at rt in 15 ml pyridine.
  • the solution was poured into a mixture of 50 g ice and 20 ml cone, hydrochloric acid, filtered and the obtained precipitate was washed twice with 20 ml ice cold HCI (4 M).
  • the precipitate was dissolved in THF and transferred to a round bottom flask and evaporated three times with toluene. This gave dexamethasone- hs (1.25 g, 100%) as a white solid.
  • Dexamethasone-hs (500 mg, 0.988 mol) was dissolved in 20 ml dry THF. N- hydroxysuccinimide (171 mg, 1.48 mmol) and EDC (200 mg, 1.29 mg) was added and the reaction was stirred overnight at rt. The solvent was removed in vacuo and the product was purified by flash chromatography (pentane-EtOAc; 1 :1 ) giving Dexamethasone-hs-NHS (376 mg, 63%) as a white solid.
  • Dexamethasone-hs-NHS were stored at -20°C and a 1 mg/ml solution in DMSO prepared freshly for each conjugation reaction.
  • Fluocinolone acetonide-hs-NHS was synthesised as for dexamethasone.
  • Antibody solutions were obtained either from purification of cell culture supernatants prepared in house (3E10B10, KN2NRY), from AbD Serotec (ED2) or from Sigma (gammaglobulins from human blood). Bovine serum albumin was obtained from Sigma.
  • Protein solutions were used in a final concentration of 1 mg/ml in either 50 mM borate buffer, pH 8.3 (ED2, hlgG, BSA) or 10 mM Na-Phosphate buffer, 140 mM NaCI, pH 6.5 (KN2/NRY, 3E10B10).
  • 50 mM borate buffer, pH 8.3 (ED2, hlgG, BSA) or 10 mM Na-Phosphate buffer, 140 mM NaCI, pH 6.5 (KN2/NRY, 3E10B10) 40 to 100 ⁇ of the 1 mg/ml Corticosteroid-NHS solution in DMSO per mg protein were added slowly to the antibody solution while gently stirring the solution on a laboratory mixer.
  • Reaction mix was then incubated for 15 minutes (for pH 8.3-buffer) or 20 hours (pH 6.5- buffer) at 25 1C on a thermomixer (Thermomixer comfort, Eppendorf AG) while gently agitating.
  • a thermomixer Thermomixer comfort, Eppendorf AG
  • pH 6.5, 140 mM NaCI were added per ml reaction mix.
  • Conjugates were sterile filtered, aliquoted into microcentrifuge tubes and stored at either 4° or 37°C for the indicated times. At sampling times they were analyzed for amount of free corticosteroid and at some dates also for protein concentration and amount of total (free + bound) Dexamethasone to check for protein precipitation or protein/drug degradation. 3. Determination of free and protein-conjugated corticosteroids by HPLC 3.1. Sample preparation
  • Protein concentration in conjugated samples was determined with the Quick Start Bradford Dye Reagent (BioRad, Denmark) according to the instructions of the manufacturer. A 2 mg/ml solution of bovine gamma globulin (Pierce, Thermo Scientific, Denmark) in PBS was used at different dilutions to obtain a linear standard curve.
  • Conjugate samples were filtered through 0.45 pm syringe filters, and 10 pg run over an Agilent Zorbax Bio Series GF-250 column in an Shimadzu 10 A HPLC System (Shimadzu, Japan) and eluted with PBS at a flow rate of 1 ml/min. Peaks were detected by absorption measurement at 280 nm by a Shimadzu SPD 10A VP detector. Run time per injection was 30 minutes. Autosampler/vial holder was set to 4 °C, analysis was at room termperature (no column heating).
  • Coupling method Dexamethasone-NHS was coupled to human gammaglobulin in 50 mM borate buffer, pH 8.3 with a coupling time of 15 minutes. 40 ⁇ Dexa-NHS solution was used per mg protein. Reaction was stopped by adding 5 mM Glycin in 50 mM borate buffer pH 8.3.
  • Fig. 3 shows the results for 37°C storage over 91 days from conjugation. Dexa release from conjugate shows a clear pH-dependency, with conjugates at low pH being more stable than those at higher pH. When stored at pH 7.8 (PBS) 100 % free drug is already achieved after 20 days.
  • PBS pH 7.8
  • Fig. 4. shows the same results for storage at 4°C over one year from conjugation.
  • the difference in dexa release when going from pH 7.4 to 6.5 is even more striking, whereas differences between pH 6.5 and 4 are not as easily visible due to low values of released dexa.
  • Figure 5 shows the same results for storage at 4°C over one year from conjugation.
  • the difference in dexa release when going from pH 7.4 to 6.5 is even more striking, whereas differences between pH 6.5 and 4 are not as easily visible due to low values of released dexa. However, these can be seen on Figure 5.
  • Protein recovery is defined as protein concentration at indicated time point in relation to concentration at time point 0.
  • Fluocinolone-Acetonide-NHS was coupled to 3E10B10 in 10 mM Na- P04, 140 mM NaCI, pH 6.5 with a coupling time of 20 hours. 50 ⁇ Fluocinolone- Acetonide-NHS solution (1mg/ml in DMSO) was used per mg protein. Reaction was stopped by adding 5 mM Glycin in 10 mM Na-P04, 140 mM NaCI, pH 6.5.
  • Fig. 7 shows the results for 4°C storage over 110 days from conjugation. The tendency is the same as for hlgG-dexamethasone conjugates, at low pH there is less fluocinolone- acetonide released than at higher pH. 3. Dexamethasone release from KN2/NRY-dexamethasone-coniugates in buffers with PH 4 to 7.8
  • Dexamethasone-NHS was coupled to KN2/NRY in 10 mM Na-P04, 140 mM NaCI, pH 6.5 with a coupling time of 20 hours. 80 ⁇ Dexa-NHS solution was used per mg protein. Reaction was stopped by adding 5 mM Glycin in 10 mM Na-P04, 140 mM NaCI, pH 6.5. Conjugates were stored at 4°C and at 37°C in the following buffers:
  • Fig. 8 shows the results for 37°C storage over 28 days from conjugation, fig. 9 and 10 for 4°C storage over 85 days.
  • Dexa release from conjugate shows the same pH- dependency as with hlgG-dexamehasone and 3E10B10-fluocinolone-acetonide- conjugates.
  • PBS pH 7.8
  • Half life of KN2/NRY conjugate at 37°C is similar to hlgG-conjugate at low pH (4.0 and 5.0, Tab. 1), but only about half of values for hlgG-conjugate at higher pH (5.5, 6.5, 7.8).
  • Half life for conjugates stored at 4°C is similar for both conjugates, though.

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Abstract

La présente invention concerne des compositions stables au stockage comprenant une fraction de liaison à un polypeptide et un agent thérapeutique, la fraction de liaison à un polypeptide étant conjuguée à l'agent thérapeutique par une liaison ester et la composition ayant un pH acide. L'invention concerne également des procédés, des utilisations et des trousses comprenant de telles compositions.
PCT/GB2012/053195 2011-12-23 2012-12-19 Nouvelles formulations Ceased WO2013093465A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105467024A (zh) * 2015-11-17 2016-04-06 宁夏泰瑞制药股份有限公司 一种泰地罗新含量检测方法
US10711032B2 (en) 2016-11-08 2020-07-14 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof
WO2021030665A1 (fr) * 2019-08-15 2021-02-18 Silverback Therapeutics, Inc. Formulations de conjugués de benzazépine et leurs utilisations
US11179473B2 (en) 2020-02-21 2021-11-23 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
US11377502B2 (en) 2018-05-09 2022-07-05 Regeneron Pharmaceuticals, Inc. Anti-MSR1 antibodies and methods of use thereof
US11491237B2 (en) 2017-05-18 2022-11-08 Regeneron Pharmaceuticals, Inc. Cyclodextrin protein drug conjugates
US11541126B1 (en) 2020-07-01 2023-01-03 Silverback Therapeutics, Inc. Anti-ASGR1 antibody TLR8 agonist comprising conjugates and uses thereof
US20230099074A1 (en) * 2015-10-06 2023-03-30 Merck Sharp & Dohme Llc Antibody drug conjugate for anti-inflammatory applications
US12070506B2 (en) 2018-01-08 2024-08-27 Regeneron Pharmaceuticals, Inc. Steroids and antibody-conjugates thereof
US12134631B2 (en) 2017-11-07 2024-11-05 Regeneron Pharmaceuticals, Inc. Hydrophilic linkers for antibody drug conjugates

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US20230099074A1 (en) * 2015-10-06 2023-03-30 Merck Sharp & Dohme Llc Antibody drug conjugate for anti-inflammatory applications
CN105467024A (zh) * 2015-11-17 2016-04-06 宁夏泰瑞制药股份有限公司 一种泰地罗新含量检测方法
US12377159B2 (en) 2016-11-08 2025-08-05 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof
US10711032B2 (en) 2016-11-08 2020-07-14 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof
US11760775B2 (en) 2016-11-08 2023-09-19 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof
US11491237B2 (en) 2017-05-18 2022-11-08 Regeneron Pharmaceuticals, Inc. Cyclodextrin protein drug conjugates
US12589101B2 (en) 2017-05-18 2026-03-31 Regeneron Pharmaceuticals, Inc. Cyclodextrin protein drug conjugates
US12134631B2 (en) 2017-11-07 2024-11-05 Regeneron Pharmaceuticals, Inc. Hydrophilic linkers for antibody drug conjugates
US12070506B2 (en) 2018-01-08 2024-08-27 Regeneron Pharmaceuticals, Inc. Steroids and antibody-conjugates thereof
US11377502B2 (en) 2018-05-09 2022-07-05 Regeneron Pharmaceuticals, Inc. Anti-MSR1 antibodies and methods of use thereof
US12497460B2 (en) 2018-05-09 2025-12-16 Regeneron Pharmaceuticals, Inc. Anti-MSR1 antibodies and methods of use thereof
WO2021030665A1 (fr) * 2019-08-15 2021-02-18 Silverback Therapeutics, Inc. Formulations de conjugués de benzazépine et leurs utilisations
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US12364768B2 (en) 2020-02-21 2025-07-22 Araris Biotech Ag Nectin-4 antibody conjugates and uses thereof
US11541126B1 (en) 2020-07-01 2023-01-03 Silverback Therapeutics, Inc. Anti-ASGR1 antibody TLR8 agonist comprising conjugates and uses thereof

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