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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07—ORGANIC CHEMISTRY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Fusion proteins which bind to human Fc receptors
• the invention relates to fusion proteins which bind to human Fc-receptors.
• the fusion proteins are initially produced as monomers, and are capable of assembly into multimers at a target site of interest.
• the invention also relates to therapeutic compositions comprising the fusion proteins, and their widespread use in the treatment of diseases.
• mAb Monoclonal antibodies
• C1 first component of complement
• FcyRs Fc gamma Receptors
• complement may be activated by IgG hexamers assembled at the cell surface. Specific noncovalent interactions between Fc segments of IgG antibodies were observed to result in the formation of ordered antibody hexamers after antigen binding on cells. The hexamers recruited and activated C1 , triggering the complement cascade. (Diebolder C.A. et al., Science 343, 1260-1263, (2014). Advances in antibody therapies have largely focussed on making the mAb more potent. In the case of enhanced effector functions this can often result in a less well tolerated drug with greater side-effects and dose-limiting toxicity. Thus, there remains a significant clinical need for improved antibody therapies with better safety profiles, which are highly effective and potent, and yet have fewer adverse side effects and low toxicity.
• the fusion proteins are initially produced as monomers, and are capable of assembly into multimers at a target site of interest.
• the fusion proteins may be used to design a new class of antibody therapeutics, that are administered as relatively inert monomers, and assemble into highly potent multimers only when they reach a desired location at a target site of interest.
• the fusion proteins may thus provide improved therapeutic compositions with greater safety, which combine enhanced efficacy and potency with fewer adverse side effects and low toxicity.
• EU numbering system is used to refer to the residues in antibody domains, unless otherwise specified. This system was originally devised by Edelman et al, 1969 and is described in detail in Kabat et al, 1987.
• position number and/or amino acid residue is given for a particular antibody isotype, it is intended to be applicable to the corresponding position and/or amino acid residue in any other antibody isotype, as is known by a person skilled in the art.
• the position number given is the position number of the residue in naturally occurring IgM or IgA, according to conventional practice in the art.
• the present invention provides a monomeric fusion protein comprising an antibody
• one or each heavy chain Fc-region is fused at its C-terminal to an antibody tailpiece
• tailpiece cysteine residue is modified to prevent disulphide bond formation.
• the tailpiece cysteine residue is deleted, substituted, or blocked with a thiol capping agent.
• the fusion protein of the present invention further comprises an antigen binding region.
• the antigen binding region may comprise any suitable antigen binding domain.
• the antigen binding region may be derived from an antibody and may for example comprise a VH and/or a VL antigen binding domain.
• the antigen binding region is selected from the group consisting of Fab, scFv, single domain antibody (dAb), and DARPin.
• a single domain antibody may be a VH, VL or VHH domain.
• the antigen binding region is fused to the N-terminus of the heavy chain Fc-region.
• the antigen binding region may be fused directly to the N-terminus of the heavy chain Fc- region.
• the fusion protein of the present invention further comprises a fusion partner.
• the term 'fusion partner' refers to an antigen, pathogen- associated molecular pattern (PAMP), drug, ligand, receptor, cytokine or chemokine.
• PAMP pathogen- associated molecular pattern
• the 'fusion partner' does not include an antibody or a variable domain derived from an antibody.
• the fusion partner is fused to the N- terminus of the heavy chain Fc-region.
• the fusion partner may be fused directly to the N-terminus of the heavy chain Fc-region.
• it may be fused indirectly by means of an intervening amino acid sequence.
• a short peptide linker or a hinge sequence may be provided between the fusion partner and the heavy chain Fc-region.
• the antibody Fc-domain component of the monomers of the present invention may be derived from any suitable species.
• the antibody Fc-domain is derived from a human Fc-domain.
• the antibody Fc-domain may be derived from any suitable class of antibody, including IgA (including subclasses lgA1 and lgA2), IgD, IgE, IgG (including subclasses lgG1 , lgG2, lgG3 and lgG4), and IgM.
• the antibody Fc-domain is derived from IgG.
• the antibody Fc-domain is derived from lgG1 .
• the antibody Fc-domain is derived from lgG2.
• the antibody Fc domain is derived from lgG3.
• the antibody Fc domain is derived from lgG4.
• the antibody Fc-domain comprises two individual polypeptide chains, each referred to as a heavy chain Fc-region.
• the two heavy chain Fc-regions dimerise to create the antibody Fc-domain. Whilst the two heavy chain Fc-regions that together form the antibody Fc domain may be different from one another it will be appreciated that these will usually be the same as one another.
• each heavy chain Fc-region comprises or consists of two or three heavy chain constant domains.
• the heavy chain Fc-region of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3) and that of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerise to create the antibody Fc domain.
• the heavy chain Fc-region may comprise heavy chain constant domains from one or more different classes of antibody, for example one, two or three different classes.
• the heavy chain Fc-region comprises CH2 and CH3 domains derived from lgG1 .
• the heavy chain Fc-region comprises CH2 and CH3 domains derived from lgG2.
• the heavy chain Fc-region comprises CH2 and CH3 domains derived from lgG3.
• the heavy chain Fc-region comprises CH2 and CH3 domains derived from lgG4.
• the CH3 domain plays a significant role in the polymerisation of fusion proteins comprising an antibody Fc-domain and a tail-piece sequence.
• the amino acid at position 355 of the CH3 domain was found to have a particularly strong effect.
• the heavy chain Fc-region comprises a CH3 domain derived from lgG1 .
• the heavy chain Fc-region comprises a CH2 domain derived from lgG4 and a CH3 domain derived from lgG1 .
• the heavy chain Fc-region comprises an arginine residue at position 355.
• the heavy chain Fc-region comprises a CH4 domain from IgM.
• the IgM CH4 domain is typically located between the CH3 domain and the tailpiece.
• the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.
• the heavy chain constant domains for use in producing a heavy chain Fc-region of the present invention may include variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid variations compared to wild type constant domains.
• the heavy chain Fc-region of the present invention comprises at least one constant domain which varies in sequence from the wild type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than the wild type constant domain. Preferably the variant constant domains are at least 50% identical or similar to a wild type constant domain.
• identity indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
• similarity indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
• leucine may be substituted for isoleucine or valine.
• Other amino acids which can often be substituted for one another include but are not limited to:
• the variant constant domains are at least 60% identical or similar to a wild type constant domain. In another example the variant constant domains are at least 70% identical or similar. In another example the variant constant domains are at least 80% identical or similar. In another example the variant constant domains are at least 90% identical or similar. In another example the variant constant domains are at least 95% identical or similar.
• one or each heavy chain Fc-region is fused at its C-terminus to an antibody tailpiece, wherein the tailpiece cysteine residue is modified to prevent disulphide bond formation.
• the antibody tailpiece of the invention may be derived from any suitable species. Antibody tailpieces are evolutionarily conserved and are found in most species, including primitive species such as teleosts. In one embodiment, the antibody tailpiece is derived from a human antibody.
• IgM and IgA occur naturally as covalent multimers of the common H 2 L 2 antibody unit.
• IgM occurs as a pentamer when it has incorporated a J-chain, or as a hexamer when it lacks a J-chain.
• IgA occurs as monomer and dimer forms.
• the heavy chains of IgM and IgA possess an 18 amino acid extension to the C-terminal constant domain, known as a tailpiece.
• This tailpiece naturally includes a cysteine residue that forms a disulphide bond with adjacent heavy chains in the polymer, and is believed to have an important role in polymerisation.
• the tailpiece also contains a glycosylation site.
• PCT/EP2015/054687 we disclosed that recombinant fusion proteins comprising an antibody Fc-domain derived from IgG and a tailpiece, are synthesised predominantly as hexamers.
• the present inventors have unexpectedly found that modification of the tailpiece cysteine residue results in fusion proteins with very unusual multimerisation properties.
• the fusion proteins of the present invention are synthesized
• the fusion proteins may be used to design a new class of antibody therapeutics, that are administered as relatively inert monomers, and assemble into highly potent multimers only when they reach a desired location at a target site of interest.
• the fusion proteins may thus provide improved therapeutic compositions with greater safety, which combine enhanced efficacy and potency with fewer adverse side effects and low toxicity.
• the monomers of the present invention are directed to a target site of interest via the Fc domain and/or, where present, the antigen binding region and/or fusion partner.
• the monomer of the present invention may comprise an antigen binding region which may bind an antigen expressed on the surface of a cell, such as a tumor cell or an immune cell, such that assembly of the monomer into a highly potent multimer may occur on the tumor cell or immune cell surface.
• Suitable antigens include HER2/neu or CD20.
• the tailpiece cysteine residue is modified to prevent disulphide bond formation.
• the cysteine residue may be modified using any any suitable method that prevents disulphide bond formation.
• the tailpiece cysteine residue is mutated.
• the tailpiece cysteine residue is deleted.
• the tailpiece cysteine residue is substituted with another amino acid residue.
• the antibody tailpiece is derived from human IgM or IgA, wherein the tailpiece cysteine residue normally found at position 575 of IgM or position 495 of IgA has been deleted or substituted with another amino acid residue.
• the tailpiece cysteine residue normally found at position 575 of IgM is substituted with a serine, threonine or alanine residue (C575S, C575T, or C575A).
• the tailpiece cysteine residue normally found at position 495 of IgA is substituted with a serine, threonine or alanine residue (C495S, C495T, or C495A).
• the tailpiece cysteine residue is blocked with a thiol capping agent.
• a thiol capping agent is a compound that reacts with sulphydryl groups in reduced cysteine residues, preventing them from forming disulphide bonds.
• thiol capping agents examples include N-ethylmaleimide, iodoacetic acid, or iodoacetamide.
• the thiol capping agent used to block the tailpiece cysteine residue is conjugated to a drug.
• the capping reaction thus effectively forms a bridge between the drug and the cysteine residue in the target protein, conjugating the drug to the tailpiece.
• the tailpiece comprises all or part of an 18 amino acid tailpiece sequence from human IgM or IgA as shown in Table 1 , wherein the cysteine residue normally found at position 575 of IgM, or position 495 of IgA, is deleted, substituted, or blocked with a thiol capping agent.
• the tailpiece may be fused directly to the C-terminus of the heavy chain Fc-region. Alternatively, it may be fused indirectly by means of an intervening amino acid sequence. For example, a short linker sequence may be provided between the tailpiece and the heavy chain Fc-region.
• the tailpiece of the present invention may include variants or fragments of the tailpiece sequences described above.
• a variant of an IgM or IgA tailpiece typically has an amino acid sequence which is identical to the described sequence in 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, or 17 of the 18 amino acid positions shown in Table 1 .
• a fragment typically comprises 8, 9, 10, 1 1 , 12, 13, 14, 15, 1 6, or 17 amino acids.
• the tailpiece may be a hybrid IgM/lgA tailpiece. Fragments of variants are also envisaged.
• Each heavy chain Fc-region of the present invention may optionally possess a native or a modified hinge region at its N-terminus.
• a native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody.
• a modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc-region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region.
• the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means the number of cysteine residues in the hinge region may be increased or decreased.
• Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility.
• the heavy chain Fc-region possesses an intact hinge region at its N-terminus.
• the heavy chain Fc-region and hinge region are derived from lgG4 and the hinge region comprises the mutated sequence CPPC (SEQ ID NO: 1 1 ).
• the core hinge region of human lgG4 naturally contains the sequence CPSC (SEQ ID NO: 1 2), compared to lgG1 which contains the sequence CPPC.
• the serine residue present in the lgG4 sequence leads to increased flexibility in this region, and therefore a proportion of molecules form disulphide bonds within the same protein chain (an intrachain disulphide) rather than bridging to the other heavy chain in the IgG molecule to form the interchain disulphide.
• a monomeric fusion protein of the invention comprises an amino acid sequence as provided in Figure 2, optionally with an alternative hinge or tailpiece sequence.
• the present invention provides a monomeric fusion protein comprising two identical polypeptide chains, each polypeptide chain comprising or consisting of the sequence given in any one of SEQ ID NOs 26 to 37.
• the present invention provides a monomeric fusion protein comprising two identical polypeptide chains, each polypeptide chain comprising or consisting of the sequence given in amino acids 6 to 222 of any one of SEQ ID NOs 26 to 29, or the sequence given in amino acids 6 to 333 of SEQ ID NOs 30 or 31 , or the sequence given in amino acids 6 to 221 of any one of SEQ ID NOs 32 to 35, or the sequence given in amino acids 6 to 332 of SEQ ID NOs 36 or 37.
• a monomeric fusion protein of the invention comprises an amino acid sequence as provided in Figure 3. Accordingly in one example the present invention provides a monomeric fusion protein comprising or consisting of an amino acid sequence given in any one of SEQ ID Nos 38 to 69.
• the monomeric fusion protein of the invention is capable of concentration-dependent assembly into multimers.
• the multimer may comprise two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more monomer units.
• Such a multimer may alternatively be referred to as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, dodecamer, etc., respectively.
• the monomeric fusion protein assembles into a hexamer.
• the present invention provides a mixture comprising a monomeric fusion protein of the invention and a multimer, said multimer comprising two or more monomer units.
• the mixture comprises a monomeric fusion protein of the invention and a hexamer. In one embodiment, the mixture comprises greater than 55% monomer, for example, greater than 65%, greater than 75%, greater than 85%, greater than 90% or greater than 95% monomer.
• the mixture is enriched for the monomeric form of the fusion protein of the invention.
• the term "enriched" means that greater than 80% of the fusion protein of the invention is present in the mixture in monomeric form, such as greater than 90% or greater than 95%. It will be appreciated that the proportion of monomer in a sample can be determined using any suitable method such as analytical size exclusion chromatography, as described herein below.
• the monomeric fusion protein of the invention is produced or formulated using a component that stabilises the monomeric form of the protein.
• the component may increase the ratio of monomer to multimer in a mixture, so that a higher proportion of the protein is present in monomeric form than would otherwise be the case.
• suitable components include pharmaceutical excipients, diluents or carriers which enable the monomeric fusion protein of the invention to be formulated at high concentration prior to administration.
• the monomeric fusion proteins of the present invention may comprise one or more mutations that alter the functional properties of the proteins, for example, binding to Fc-receptors such as leukocyte receptors, complement or FcRn.
• the present invention also provides an isolated DNA sequence encoding a polypeptide chain of the present invention, or a component part thereof.
• the DNA sequence may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof.
• DNA sequences which encode a polypeptide chain of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of a polypeptide chain may be synthesised as desired from the determined DNA sequences or on the basis of the corresponding amino acid sequences.
• a monomeric fusion protein of the invention is encoded by a DNA sequence as provided in Figure 3. Accordingly in one example the present invention provides a DNA sequence given in any one of SEQ ID Nos 38 to 69.
• the present invention also relates to a cloning or expression vector comprising one or more DNA sequences of the present invention. Accordingly, provided is a cloning or expression vector comprising one or more DNA sequences encoding a
• polypeptide chain of the present invention or a component part thereof.
• a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding a monomeric fusion protein of the present invention.
• Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the monomeric fusion protein of the present invention.
• Bacterial for example E. coli, and other microbial systems such as Saccharomyces or Pichia may be used or eukaryotic, for example mammalian, host cell expression systems may also be used.
• Suitable mammalian host cells include CHO cells.
• Suitable types of Chinese hamster ovary (CHO cells) for use in the present invention may include CHO and CHO-K1 cells, including dhfr- CHO cells, such as CHO-DG44 cells and CHO-DXB1 1 cells, which may be used with a DHFR selectable marker, or CHOK1 -SV cells which may be used with a glutamine synthetase selectable marker.
• Other suitable host cells include NSO cells.
• the present invention also provides a process for the production of a monomeric fusion protein according to the present invention, comprising culturing a host cell containing a vector of the present invention under conditions suitable for expression of the monomeric fusion protein, and isolating and optionally purifying the monomeric fusion protein.
• the monomeric fusion proteins of the present invention are expressed at good levels from host cells. Thus the properties of the monomeric fusion protein are conducive to commercial processing.
• the monomeric fusion proteins of the present invention may be made using any suitable method.
• the monomeric fusion protein of the invention may be produced under conditions which minimise aggregation. In one example, aggregation may be minimised by the addition of preservative to the culture media, culture supernatant, or purification media. Examples of suitable preservatives include ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), or acidification to below pH 6.0.
• EDTA ethylenediaminetetraacetic acid
• EGTA ethyleneglycoltetraacetic acid
• a process for purifying a monomeric fusion protein of the present invention comprising the steps: performing anion exchange chromatography in non-binding mode such that the impurities are retained on the column and the monomeric fusion protein is eluted.
• the purification employs affinity capture on an FcRn, FcyR or C- reactive protein column.
• the purification employs protein A.
• Suitable ion exchange resins for use in the process include Q.FF resin (supplied by GE-Healthcare).
• the step may, for example be performed at a pH about 8.
• the process may further comprise an initial capture step employing cation exchange chromatography, performed for example at a pH of about 4 to 5, such as 4.5.
• the cation exchange chromatography may, for example employ a resin such as CaptoS resin or SP sepharose FF (supplied by GE-Healthcare).
• the monomeric fusion protein can then be eluted from the resin with an ionic salt solution such as sodium chloride, for example at a concentration of 200mM.
• the chromatography step or steps may include one or more washing steps, as appropriate.
• the purification process may also comprise one or more filtration steps, such as a diafiltration step.
• the monomeric fusion proteins of the invention can be purified according to molecular size, for example by size exclusion chromatography.
• a purified monomeric fusion protein according to the invention in substantially purified from, in particular free or substantially free of endotoxin and/or host cell protein or DNA.
• Purified form as used herein is intended to refer to at least 90% purity, such as 91 , 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.
• Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU per mg protein product or less, such as 0.5 or 0.1 EU per mg product.
• Substantially free of host cell protein or DNA is generally intended to refer to a host cell protein and/or DNA content of 400 ⁇ g per mg of protein product or less, such as 100 ⁇ g per mg product or less, in particular 20 ⁇ g per mg product.
• the present invention also provides a pharmaceutical or diagnostic composition comprising a monomeric fusion protein of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier.
• the present invention also provides a process for preparation of a pharmaceutical or diagnostic composition comprising adding and mixing the monomeric fusion protein of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.
• the pharmaceutical composition comprises a component that stabilises the monomeric form of the protein or increases the ratio of monomer to multimer in a mixture.
• the monomeric fusion protein may be the sole active ingredient in the
• composition or may be accompanied by other active ingredients including antibody ingredients or non-antibody ingredients such as other drug molecules.
• compositions suitably comprise a therapeutically effective amount of the monomeric fusion protein of the invention.
• therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
• the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• the precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug
• compositions may be conveniently presented in unit dose forms containing a predetermined amount of a monomeric fusion protein of the invention per dose. Compositions may be administered individually to a patient or may be administered in combination (e.g. simultaneously, sequentially or separately) with other agents, drugs or hormones.
• the frequency of dose will depend on the half-life of the monomeric fusion protein and the duration of its effect. If the monomeric fusion protein has a short half-life (e.g. 2 to 10 hours) it may be necessary to give one or more doses per day.
• the monomeric fusion protein has a long half-life (e.g. 2 to 15 days), and/or long lasting pharmacodynamic effects, it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 months.
• a long half-life e.g. 2 to 15 days
• the dose is delivered bi-weekly, i.e. twice a month.
• Half-life as employed herein is intended to refer to the duration of the monomeric fusion protein in circulation, for example in serum or plasma.
• Pharmacodynamics as employed herein refers to the profile and, in particular, duration of the biological action of the monomeric fusion protein.
• the pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.
• Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
• Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
• Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions.
• auxiliary substances such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions.
• the therapeutic suspensions or solution formulations can also contain one or more excipients.
• Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres.
• the formulation will generally be provided in a substantially sterile form employing sterile manufacture processes. This may include production and sterilization by filtration of the buffered solvent/solution used for the formulation, aseptic suspension of the protein in the sterile buffered solvent solution, and dispensing of the
• Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion.
• the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents.
• the monomeric fusion protein may be in the form of nanoparticles. Alternatively, the monomeric fusion protein may be in dry form, for reconstitution before use with an appropriate sterile liquid.
• compositions of the invention can be administered directly to the subject.
• the subjects to be treated can be animals. However, in one or more embodiments the compositions are adapted for administration to human subjects.
• compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes.
• routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes.
• routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranas
• compositions may be prepared as injectables, either as liquid solutions or
• compositions Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
• Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
• the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule
• the active ingredient in the composition will be a protein molecule. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the protein from degradation but which release the protein once it has been absorbed from the gastrointestinal tract.
• administrations including inhalation.
• a monomeric fusion protein of the invention for the manufacture of a medicament for the treatment of cancer.
• the cancer is skin cancer, such as melanoma.
• the cancer is Leukemia.
• the cancer is glioblastoma, medulloblastoma or neuroblastoma.
• the cancer is a neuroendocrine cancer.
• the cancer is Hodgkin's or non-Hodgkins lymphoma.
• ADAM Disseminated Encephalomyelitis
• Amyloidosis ANCA-associated vasculitis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency , Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease,
• GPA Polyangiitis
• Interstitial cystitis Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome, Kuttner's tumour, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Mediastinal fibrosis, Meniere's disease, Microscopic polyangiitis, Mikulicz's syndrome, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multifocal fibrosclerosis, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Ormond's disease (retroperitoneal fibrosis), Palindromic rheumatism, PANDAS (Pediatric Autoimm
• thrombocytopenic purpura TTP
• Tolosa-Hunt syndrome TTP
• Transverse myelitis Ulcerative colitis
• Undifferentiated connective tissue disease UCTD
• Uveitis Uveitis
• Vasculitis Vesiculobullous dermatosis
• Vitiligo Waldenstrom Macroglobulinaemia
• Warm idiopathic haemolytic anaemia and Wegener's granulomatosis now termed Granulomatosis with Polyangiitis (GPA).
• the autoimmune disease is selected from immune thrombocytopenia (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), Kawasaki disease and Guillain-Barre syndrome (GBS).
• the monomeric fusion proteins of the invention are employed in the treatment or prophylaxis of epilepsy or seizures.
• the monomeric fusion proteins and fragments according to the disclosure are employed in the treatment or prophylaxis of multiple sclerosis.
• the monomeric fusion protein according to the present disclosure may be employed in treatment or prophylaxis.
• the monomeric fusion protein of the present invention may also be used in diagnosis, for example in the in vivo diagnosis and imaging of disease states involving Fc-receptors, such as B-cell related lymphomas.
• Example amino acid sequences of a polypeptide chain of a monomeric fusion protein In each sequence, the tailpiece sequence is underlined, and any mutations are shown in bold and underlined. The hinge is in bold. In constructs comprising a CH4 domain from IgM, this region is shown in italics.
• Figure 3 In constructs comprising a CH4 domain from IgM, this region is shown in italics.
• Example amino acid and DNA sequences for antibody fusion proteins of the invention are underlined.
• HMWS high molecular weight species
• DNA sequences were assembled using standard molecular biology methods, including PCR, restriction-ligation cloning, point mutagenesis (Quikchange) and Sanger sequencing.
• Expression constructs were cloned into expression plasmids (pNAFL, pNAFH) suitable for both transient and stable expression in CHO cells.
• suitable expression vectors include pCDNA3 (Invitrogen). Diagrams showing example amino acid sequences of a polypeptide chain of a monomeric fusion protein are provided in Figure 2. In each sequence, the tailpiece sequence is underlined, and any mutations are shown in bold and underlined. The hinge is in bold. In constructs comprising a CH4 domain from IgM, this region is shown in italics.
• Example 2 Expression Small scale expression was performed using 'transient' expression of HEK293 or CHO cells transfected using lipofectamine or electroporation. Cultures were grown in shaking flasks or agitated bags in CD-CHO (Lonza) or ProCHO5 (Life Technologies) media at scales ranging from 50 - 2000ml for 5-10 days. Cells were removed by centrifugation and culture supernatants were stored at 4°C until purified.
• Preservatives were added to some cultures after removal of cells.
• Example 3 Purification and analysis Proteins were purified from culture supernatants after checking / adjusting pH to be >6.5, by protein A chromatography with step elution using a pH3.4 buffer. Eluate was immediately neutralised to ⁇ pH7.0 using 1 M Tris pH8.5.
• Samples were concentrated in 0.1 M sodium citrate buffer pH7.5 using a pressure stirred cell, and 10kDa cut-off membrane to >100mg/ml. Samples were then diluted in PBS to a range of different final concentrations as shown in Table 3, before SE- HPLC analysis of multimeric form as described below. Endotoxin was tested using the limulus amoebocyte lysate (LAL) assay. Samples used in assays were
• Proteins were analysed using size exclusion HPLC, the column used was 15 ml TSKgel -G3000SW (Tosoh) on system Agilent 1 100 Series. The mobile phase was 0.2 M sodium phosphate, pH7.0, flow rate 1 ml/minute, 50 ⁇ g protein injected. Signal was detected using a UV absorbance detector at 280 nm wavelength.
• uPLC Proteins were analysed using size exclusion HPLC, the column used was 2.5ml BEH 200 (Waters) on system Agilent 1 100 Series. The mobile phase was 0.2 M sodium phosphate, pH7.0, flow rate 0.4 ml/minute, 2.5 and 5 ⁇ g protein injected. Signal was detected using a Fluorescence detector Excitation: 350 nm, Emission: 390nm.
• Proteins were analysed using size exclusion HPLC, the column used was 24ml Superdex 200 10/300 (GE) on system Agilent 1 100 Series.
• the mobile phase was 10mM HEPES, 100mM NaCI pH7.5, flow rate 0.5 ml/minute, 100 ⁇ g protein injected.
• Signal was detected using a UV absorbance detector at 280 nm wavelength and additional refractive index detector (Viscotek VE3580) and MALS detector (Viscotech SEC-MALS 20, Malvern)
• Control protein Hinge-Fc (no tailpiece):
• Control proteins lacking the modified tailpiece are unable to assemble into hexamers, even at the highest concentrations tested.
• Proteins were concentrated by centrifugation using Amicon Ultra-15 Centrifugal Filter Units. Samples were centrifuged at 4000 RPM until desired concentration was reached. Proteins were analysed using size exclusion HPLC, the column used was 15 ml TSKgel -G3000SW (Tosoh) on system Agilent 1 100 Series. The mobile phase was 0.2 M sodium phosphate, pH7.0, flow rate 1 ml/minute, 50 ⁇ g protein injected. Signal was detected using a UV absorbance detector at 280 nm wavelength.
• CDC assay The biological activity of the modified antibodies was examined using a complement dependent cytotoxicity assay.
• Target cells 50 x 10 5
• Opsonised target cells were incubated with normal human serum at a final concentration of 30% (42 ⁇ added) and incubated in a 37°C incubator for 30 minutes.
• Cell lysis measured as a fraction of PI+ cells determined by BD FACSCalibur flow cytometer.
• Graphs were plotted using GraphPad Prism software. Results for a fusion protein comprising the anti-CD20 antibody rituximab are shown in Figure 5.
• the graphs show complement killing of CD20 positive Raji cells and CD20 positive Ramos cells.
• the results demonstrated that rituximab shows enhanced CDC when modified with the tailpiece of the invention.

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