WO2016009232A1 - Proteines polymeres et utilisations de celles-ci - Google Patents

Proteines polymeres et utilisations de celles-ci Download PDF

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WO2016009232A1
WO2016009232A1 PCT/GB2015/052098 GB2015052098W WO2016009232A1 WO 2016009232 A1 WO2016009232 A1 WO 2016009232A1 GB 2015052098 W GB2015052098 W GB 2015052098W WO 2016009232 A1 WO2016009232 A1 WO 2016009232A1
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seq
protein according
immunoglobulin
polymeric
polymeric protein
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Richard PLEASS
Patricia BLUNDELL
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Liverpool School of Tropical Medicine
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Liverpool School of Tropical Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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

  • the present invention relates to polymeric proteins.
  • the invention also relates to the medical uses of such proteins, in particular in the prevention or treatment of autoimmune diseases such as idiopathic thrombocytopenia.
  • the invention further relates to methods of treatment using polymeric proteins, and to pharmaceutical compositions comprising polymeric proteins.
  • ADs Autoimmune diseases
  • IVIG purified immunoglobulin G
  • IDP Food and Drug Administration
  • Kawasaki disease Guillain-Barre
  • dermatomyositis dermatomyositis
  • chronic inflammatory demyelinating polyneuropathy idiopathic thrombocytopenia (ITP)
  • IDP idiopathic thrombocytopenia
  • Kawasaki disease Guillain-Barre
  • dermatomyositis chronic inflammatory demyelinating polyneuropathy
  • the invention provides a polymeric protein comprising two or more polypeptide monomer units, each monomer unit comprising two chimeric protein chains; wherein each chimeric polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
  • each immunoglobulin G heavy chain constant region comprises a cysteine residue which is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomer unit;
  • each chimeric protein chain also comprises a modified immunoglobulin M tailpiece region
  • amino acid sequence of each chimeric polypeptide monomer comprises an alteration of the primary structure as compared to the native sequences from which the immunoglobulin G heavy chain constant region or immunoglobulin M tailpiece region are derived, and the alteration changes the number of glycosylation sites in a manner that promotes polymerisation.
  • the invention provides a polymeric protein according to the first aspect of the invention for use as a medicament.
  • the invention provides a polymeric protein according to the first aspect of the invention for use as a medicament in the prevention or treatment of an autoimmune or inflammatory disease.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polymeric polypeptide according to the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a method of preventing or treating an autoimmune or inflammatory disease, the method comprising providing a subject in need of such treatment with a therapeutically effective amount of a polymeric protein of the invention.
  • the invention provides a method of intravenous immunoglobulin (IVIG) therapy, the method comprising providing a subject in need of such treatment with a therapeutically effective amount of a polymeric protein of the invention.
  • IVIG intravenous immunoglobulin
  • the present invention is based upon the inventors' surprising finding that alterations to the number of glycosylation sites within the primary sequence of chimeric proteins produced by combining sequences of IgG and IgM are able to significantly impact upon the propensity of these proteins to polymerise.
  • altering the number of glycosylation sites within such proteins is able to promote polymerisation. This promotion of polymerisation may be manifest in the generation of tetrameric, hexameric, and even dodecameric forms of the proteins.
  • the sequence of the tailpiece of IgM has previously been modified in a manner that prevented attachment of glycan at Asn563 of IgM.
  • This modification resulted in enhanced polymer formation, evident in an increase in the number of hexamers produced by such modified proteins as compared to the number of pentamers.
  • the inventors were surprised to find that the extent of polymerisation in polypeptides of the invention far exceeded that which might be expected based upon previously available information. Indeed in illustrative examples of the polypeptides of the invention >95% of the protein is secreted as a discrete dodecameric species, with no hexamers, pentamers or tetramers observed.
  • the inventors believe that the production of immunoglobulin-based dodecamers, and certainly such production at the high levels observed in the present case, is a property that is, as yet, unique to the polymeric proteins of the invention.
  • the polypeptides of the invention may incorporate further variations from the native sequences of the IgG and/or IgM from which they are derived.
  • a suitable polymeric protein of the invention may utilise IgG and/or IgG derived sequences that share at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the relevant native IgG or IgM sequence from which they are derived.
  • the immunoglobulin G heavy chain constant regions employed in the polymeric proteins of the invention are derived from an immunoglobulin selected from the group consisting of: lgG1 ; lgG2; lgG3; and lgG4.
  • the immunoglobulin G heavy chain constant regions may be derived from lgG1.
  • the chimeric protein monomers may comprise residues 221 to 447 of human lgG1 combined with residues 561 to 575 of the tailpiece of human IgM.
  • immunoglobulin sequences that make up the chimeric proteins naturally include two sites that undergo N-linked glycosylation, namely the asparagine corresponding to residue 297 of the Fc of lgG1 (Asn297) and the asparagine corresponding to residue 563 of IgM (Asn563).
  • a polymeric protein consisting of chimeric protein chains having the sequence set out in SEQ ID NO: 1 will not constitute a polymeric protein of the invention, since it will not incorporate the requisite alteration in primary structure.
  • the IgG and IgM-derived portions of the chimeric proteins may be joined via a linker sequence.
  • the inventors have found that a 6 amino acid linker sequence Leu-Val-Leu- Gly-Pro-Pro inserted between residue K447 of IgG and residue L450 of the tailpiece facilitates polymerisation, since it allows cysteine residues in the tailpiece to associate more closely with cysteine residues in adjacent monomers, thereby increasing the formation of covalent bonds.
  • the polypeptides of the invention may incorporate a six amino acid linker between the IgG and IgM derived sequences.
  • the six amino acid linker may comprise Leu- Val-Leu-Gly-Pro-Pro. This linker is present in the reference chimeric protein, where it facilitates the involvement of Cys248 of SEQ ID NO: 1 (corresponding to Cys575 in IgM) in covalent bonds.
  • the alteration of the primary structure that changes the number of glycosylation sites comprises disruption of the glycosylation site corresponding to Asn297 in the Fc of lgG1 , or disruption of the glycosylation site corresponding to Asn563 of IgM.
  • Such disruption may decrease the total number of Disruption of the glycosylation sites may be achieved by substitution of any of the residues that are required in the asparagine-X-serine/threonine motif at which N-glycosylation occurs. It will be appreciated that any of these residues may be replaced with any residue (either naturally occurring or non-naturally occurring) that disrupts the motif.
  • the asparagine residue (Asn) may be substituted with an alanine residue (Ala).
  • the alteration of the primary structure that changes that number of glycosylation sites in a manner that promotes polymerisation may comprise the addition of a further glycosylation site not found in the native sequences from which the fusion protein has been derived.
  • polypeptides of the invention include those, such as SEQ ID NOs: 5, 6, and 7 that incorporate the introduction of an Asn residue at the N-terminal (a change to residue 1 of SEQ ID NO: 1 also referred to herein as D001 N). This causes the formation of a new glycosylation motif that is not found in SEQ ID NO: 1.
  • references to polymeric proteins that include a disruption of the glycosylation site corresponding to Asn297 in the Fc of lgG1 , or disruption of the glycosylation site corresponding to Asn563 of IgM, should be taken as limited to those polymeric proteins in which only one of these glycosylation sites has been disrupted. Such a definition does not encompass polymeric proteins in which both the glycosylation site corresponding to Asn297 in the Fc of lgG1 and the glycosylation site corresponding to Asn563 of IgM have been disrupted.
  • the alteration may comprise disruption of the glycosylation site corresponding to Asn77 of SEQ ID NO: 1 or of the glycosylation site corresponding to Asn236 of SEQ ID NO: 1.
  • disruption may comprise disruption of the glycosylation site corresponding to Asn77 of SEQ ID NO: 1 or of the glycosylation site corresponding to Asn236 of SEQ ID NO: 1.
  • polypeptides in which both the glycosylation site at Asn297 of lgG/Asn77 of SEQ ID NO: 1 and the glycosylation site at Asn563 of IgM/Asn 236 of SEQ ID NO: 1 have been disrupted do not polymerise at all, and so do not constitute polymeric proteins of the invention.
  • the alteration of the primary structure comprises an alteration at a residue corresponding to Asn563 of IgM.
  • this alteration of the primary structure comprises an alteration at a residue corresponding to Asn236 of SEQ ID NO:1.
  • polymeric proteins comprising this alteration have particularly surprising and beneficial properties. In particular, these proteins exhibit much higher levels of polymerisation than have previously been described in the scientific literature.
  • the polymeric proteins of SEQ ID NO:3 and SEQ ID NO:7 both exhibit a tendency to form dodecamers.
  • dodecamers constitute >99% of the total polymers present, without significant quantities of other polymeric intermediaries (e.g. hexamers, pentamers as seen with IgM). This remarkable level of homogeneity is particularly advantageous for therapeutic applications, where a homogeneous product is important to ensure consistency of dosing, and hence of therapeutic activity.
  • SEQ ID NO:3 differs from SEQ ID NO:1 only in respect of the substitution of Asn236 with an alanine residue
  • SEQ ID NO:7 is an example of a polymeric protein of the invention in which a further glycosylation site has been added, specifically by the introduction of an asparagine residue at the N-terminal (corresponding to residue 1 of SEQ ID NO: 1) which yields an additional glycosylation site.
  • a polymeric protein according to this aspect of the invention may comprise a sequence selected from the group consisting of: SEQ ID NO:3; and SEQ ID NO:7.
  • a polymeric protein according to this embodiment of the invention comprises SEQ ID NO:3, which is to say that the polymeric protein incorporates the sequence of SEQ ID NO:3, and optionally further amino acid residues.
  • a polymeric protein according to this embodiment of the invention comprises SEQ ID NO:7. As with the preceding paragraph, this definition should be taken as encompassing a polymeric protein that incorporates the sequence of SEQ ID NO:7, and optionally further amino acid residues.
  • a polymeric protein according to this aspect of the invention may consist of a sequence selected from the group consisting of: SEQ ID NO:3; and SEQ ID NO:7.
  • a polymeric protein of the invention consists of monomer units of SEQ ID NO:3.
  • a polymeric protein of the invention consists of monomer units of SEQ ID NO:7.
  • Polymeric proteins of the invention may alternatively comprise an alteration at a residue corresponding to Asn297 in the Fc of IgGl In the case of a polymeric protein based upon SEQ ID NO: 1 , this comprises an alteration at a residue corresponding to Asn77 of SEQ ID NO: 1.
  • SEQ ID NO:2 differs from SEQ ID NO: 1 only in that Asn77 has been substituted by an alanine residue.
  • SEQ ID NO:6 is a further example of a polymeric protein of the invention in which a further glycosylation site has been added. Once again (as with SEQ ID NO:7 discussed above), this has been achieved by the introduction of an asparagine residue at the N-terminal (corresponding to residue 1 of SEQ ID NO:1) which yields an additional glycosylation site not found in the corresponding portion of native IgGl
  • Both proteins made up of SEQ ID NO:2 and those made up of SEQ ID NO: 6 exhibit high levels of polymerisation - with both proteins being found predominantly in the hexameric form (see the Examples).
  • a polymeric protein according to this aspect of the invention may comprise a sequence selected from the group consisting of: SEQ ID NO:2; and SEQ ID NO:6.
  • a polymeric protein according to this embodiment of the invention comprises SEQ ID NO:2.
  • a polymeric protein according to this embodiment of the invention may comprise SEQ ID NO:6.
  • a polymeric protein according to this aspect of the invention may consist of a sequence selected from the group consisting of: SEQ ID NO: 2; and SEQ ID NO:6.
  • a polymeric protein of the invention consists of monomer units of SEQ ID NO:2.
  • a polymeric protein of the invention consists of monomer units of SEQ ID NO:6.
  • the alteration of the primary structure that changes that number of glycosylation sites in a manner that promotes polymerisation may comprise the addition of a further glycosylation site not found in the native sequences from which the fusion protein has been derived. While in SEQ ID NO:6 and SEQ ID NO:7 such a change is found in combination with a disruption of one or other of the naturally occurring glycosylation sites in the IgG or IgM sequences from which the fusion protein is derived, in a suitable embodiment the addition of a further glycosylation site in this manner may be the only alteration, and may still serve to promote polymerisation.
  • the fusion protein of SEQ ID NO:5 retains both Asn77 and Asn236 of SEQ ID NO: 1 , while adding a further glycosylation site at the N-terminus of the fusion protein through the substitution of the naturally occurring aspartic acid residue with an asparagine residue.
  • This alteration of the primary sequence is again associated with the promotion of polymerisation, and proteins made up of SEQ ID NO:5 are found in hexameric form.
  • substitution of aspartic acid at position 1 of SEQ ID NO: 1 with an asparagine residue causes the addition of a glycosylation site in the hinge region of the immunoglobulin G heavy chain constant region.
  • Glycosylation of the hinge region of antibodies is not found in nature, and it would be expected that this would interfere with disulphide bond formation. Since disulphide bond formation is necessary for the production of homodimers associated with formation of the Fc receptor binding portion, it is surprising to find that these polymeric proteins of the invention in which glycosylation occurs within the hinge region are biologically active.
  • polymeric proteins of the invention also encompass fragments or variants of the specific polypeptide monomer units set out in SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:8 that share the biological activity of these "parent" polypeptides.
  • the fragments or variants should share at least the increased tendency towards polymerisation of the parent polypeptide from which they are derived.
  • these fragments or variants may also share the activities discussed further below that make the parent polymeric proteins suitable for use in therapeutic applications.
  • a fragment of a polypeptide monomer unit set out in SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:6; or SEQ ID NO:8 should include the alteration of the primary structure found in the parent polypeptide.
  • a variant of a polypeptide monomer unit set out in SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:6; or SEQ ID NO:8 should also include the alteration of the primary structure found in the parent polypeptide.
  • a variant may share 70% or more identity with the parent polypeptide; 80% or more identity with the parent polypeptide; 90% or more identity with the parent polypeptide; 95% or more identity with the parent polypeptide; 96% or more identity with the parent polypeptide; 97% or more identity with the parent polypeptide; 98% or more identity with the parent polypeptide; or even 99% or more identity with the parent polypeptide.
  • a variant may differ from the parent polypeptide by 1 % or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 20% or more, or even 30% or more with reference to sequence identity.
  • a suitable example of the variants considered above is a polymeric protein of the invention in which the position of one or more of the glycosylation sites set out in the polypeptide sequences of SEQ ID NOs: 2, 3, 5, 6, or 7 is "moved" as compared to these illustrative sequences.
  • a variant in accordance with this embodiment may have the Asn normally found at position 77 of SEQ ID NO: 1 moved to a position corresponding to any of residues 72, 73, 74, 75, 76, 78, 79, 80, 81 , or 82 (with the other associated residues of the glycosylation site undergoing a corresponding move).
  • such a variant may have the Asn at position 236 of SEQ ID NO: 1 moved to a position corresponding to any of residues 231 , 232, 233, 234, 235, 237, 238, 239, 240, or 241 (with the other associated residues of the glycosylation site undergoing a corresponding move).
  • a further alternative or additional modification in a variant in accordance with this embodiment of the invention may involve the inclusion of a glycosylation site in which the Asn residue is positioned at a residues corresponding to any of 2, 3, 4, 5, or 6 of SEQ ID NO:1.
  • glycan processing is affected because the peptide backbone constrains the molecular space available for glycan to be accommodated. This may also affect the nature of the glycans attached, such that these modifications may favour the addition of oligomannose type glycans over complex-types, and thus also affect interactions with effectors, as considered elsewhere in this disclosure.
  • the proteins of the invention also have biological activities that go beyond their ability to exhibit high levels of polymerisation. It is these properties, and their therapeutic applications, that give rise to the second, third, fourth, fifth, and sixth aspects of the invention.
  • polymeric proteins of the invention indicate that that they will be able to influence biological activities in therapeutically effective ways. This is reflected in the second aspect of the invention, which provides a polymeric protein in accordance with the first aspect of the invention for use as a medicament.
  • the third and fifth aspects of the invention respectively relate to medical use of the polymeric proteins of the invention in the prevention or treatment of an autoimmune or inflammatory disease, and methods of preventing or treating an autoimmune or inflammatory disease, in which a subject in need of such treatment is provided with a therapeutically effective amount of a polymeric protein.
  • Suitable autoimmune or inflammatory diseases for treatment include those that are treatable with intravenous immunoglobulin (IVIG).
  • autoimmune cytopenias Guillain-Barre syndrome, myasthenia gravis, anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis
  • autoimmune cytopenias Guillain-Barre syndrome, myasthenia gravis
  • anti-Factor VIII autoimmune disease dermatomyositis, vasculitis, and uveitis
  • autoimmune cytopenias See, van der Meche FG et al, Lancet i, 406 (1984); Sultan Y et al, Lancet ii, 765 (1984); Dalakas MC et al, N. Engl. J. Med. 329, 1993 (1993); Jayne DR et al, Lancet 337, 1137 (1991); LeHoang P et al, Ocul. Immunol. Inflamm.
  • IVIG is typically used to treat idiopathic thrombocytopenic purpura (ITP), Kawasaki disease, Guillain-Barre syndrome and chronic inflammatory demyelinating polyneuropathy (Orange et al, 2006, J Allergy Clin Immunol 117: S525-53). IVIG is also increasingly used to treat a diverse array of other autoimmune diseases which are non-responsive to mainstay therapies, including arthritis, diabetes, myositis, Crohn's colitis and systemic lupus erythematosus.
  • Autoimmune or inflammatory diseases suitable for treatment include autoimmune cytopenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, asthma, Kawasaki disease, Guillain-Barre syndrome, Stevens-Johnson syndrome, Crohn's colitis, diabetes, chronic inflammatory demyelinating polyneuropathy myasthenia gravis, anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, uveitis or Alzheimer's disease.
  • Conditions to be treated may include an inflammatory disease with an imbalance in cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic relapsing autoimmune, inflammatory, or infectious disease or process.
  • inflammatory component such as Amyotrophic Lateral Sclerosis, Huntington's Disease, Alzheimer's Disease, Parkinson's Disease, Myocardial Infarction, Stroke, Hepatitis B, Hepatitis C, Human Immunodeficiency Virus associated inflammation, adrenoleukodystrophy, and epileptic disorders especially those believed to be associated with postviral encephalitis including Rasmussen Syndrome, West Syndrome, and Lennox- Gastaut Syndrome.
  • Conditions to be treated may be hematoimmunological diseases, e.g., Idiopathic Thrombocytopenic Purpura, alloimmune/autoimmune thrombocytopenia, Acquired immune thrombocytopenia, Autoimmune neutropenia, Autoimmune hemolytic anemia, Parvovirus B19-associated red cell aplasia, Acquired antifactor VIII autoimmunity, acquired von Willebrand disease, Multiple Myeloma and Monoclonal Gammopathy of Unknown Significance, Aplastic anemia, pure red cell aplasia, Diamond-Blackfan anemia, hemolytic disease of the newborn, Immune-mediated neutropenia, refractoriness to platelet transfusion, neonatal post-transfusion purpura, hemolytic uremic syndrome, systemic Vasculitis, Thrombotic thrombocytopenic purpura, or Evan's syndrome.
  • Idiopathic Thrombocytopenic Purpura e.g., Idiopathic
  • a neuroimmunological disease may be treated, e.g., Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgM demyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated with anti- GM1 antibodies, Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis, Myelopathy especially associated with Human T-cell lymphotropic virus-1 , Autoimmune Diabetic Neuropathy, or Acute Idiopathic Dysautonomic Neuropathy or Alzheimer's disease.
  • Guillain-Barre syndrome Chronic Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic I
  • a rheumatic disease may be treated, e.g., Kawasaki's disease, Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis, Dermatomyositis, Antiphospholipid syndromes, Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis, Raynaud's, CREST syndrome or Uveitis.
  • Kawasaki's disease e.g., Kawasaki's disease, Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis, Dermatomyositis, Antiphospholipid syndromes, Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis, Raynaud's, CREST syndrome or Uveitis.
  • a dermatoimmunological disease may be treated, e.g., Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune skin blistering diseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigus foliaceus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, Atopic dermatitis or steroid dependent Atopic dermatitis.
  • Epidermal Necrolysis e.g., Epidermal Necrolysis, Gangrene, Granuloma
  • Autoimmune skin blistering diseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigus foliaceus
  • Vitiligo Streptococcal toxic shock syndrome
  • Scleroderma systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, Atopic dermatitis or steroid dependent Atopic dermatitis.
  • a musculoskeletal immunological disease may be treated, e.g., Inclusion Body Myositis, Necrotizing fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorin (BJ antigen) Myopathy, Paraneoplastic Necrotic Myopathy, X-linked Vacuolated Myopathy, Penacillamine-induced Polymyositis, Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy.
  • a gastrointestinal immunological disease may be treated, e.g., pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis or sclerosing cholangitis.
  • the disease can be, for example, post-infectious disease inflammation, Asthma, Type 1 Diabetes mellitus with anti-beta cell antibodies, Sjogren's syndrome, Mixed Connective Tissue Disease, Addison's disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferative glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolyarterits, Churg-Strauss syndrome, Polyarteritis nodosa or Multisystem organ failure.
  • ITP idiopathic thrombocytopenic purpura
  • the invention provides a method of intravenous immunoglobulin (IVIG) therapy, the method comprising providing a subject in need of such treatment with a therapeutically effective amount of a polymeric protein of the invention.
  • IVIG treatment is of benefit to many patients subject to autoimmune or inflammatory diseases.
  • Polymeric proteins of the invention for use in IVIG may be selected on the basis of increased avidity of binding to inhibitory glycan- and Fc-receptors involved in controlling pro-inflammatory responses at the expense of binding pro-inflammatory glycan receptors that lead to enhanced clearance and potential adverse events.
  • Polymeric proteins of the invention including an additional glycosylation site at the position corresponding to residue 1 of SEQ ID NO: 1 exhibit decreased complement activation and this may be desirable although removal of complement binding sites e.g. Lys322 to Ala could also be contemplated if complement activation is not desirable in the final medicament.
  • the fourth aspect the invention provides pharmaceutical compositions comprising a polymeric polypeptide of the invention and a pharmaceutically acceptable carrier.
  • Suitable additional constituents of that may be included in a pharmaceutical composition of the invention include excipients, diluents, buffering agents and preservatives. Examples of such additional constituents will be apparent to those of skill in the art on consideration of the use to which the composition is to be put, and the desired route of administration. The following paragraphs provide details of certain considerations that may be used in determining which polymeric proteins of the invention are suitable for use in the various therapeutic applications set out above.
  • polymeric proteins of the invention suitable for use in accordance with these embodiments will further comprise at least one therapeutic moiety associated with the monomer units.
  • the polymeric proteins of the invention may comprise at least one therapeutic moiety associated with each of the chimeric protein chains.
  • the therapeutic moiety may be fused to, or otherwise associated with, the chimeric protein chains. Since the polymeric proteins of the invention are able to produce dodecamers comprising 24 chimeric protein chains, if each of these is associated with a therapeutic moiety then very large numbers of such moieties may be provided by the molecules of the invention. Furthermore, this can be achieved without loss of binding to critical receptors e.g. DC-SIGN and complement.
  • Therapeutic moieties that may be attached to the polymeric proteins of the invention include drugs, toxins and other cleavable payloads.
  • the polymeric proteins of the invention also lend themselves to use in improved vaccines.
  • the large numbers of antigen binding sites present in the polymeric proteins of the invention means that they are able to increase the number of antigens delivered to dendritic cells (DCs) - thus increasing the depot effect of adjuvants.
  • the binding of polymeric proteins of the invention (such as those comprising SEQ ID NO:3, also referred to as N236A) to monocytes and DC-SIGN illustrates their properties that are highly attractive with respect to vaccine use.
  • the capacity of the polymeric proteins of the invention to bind to the FcRn may be altered by modification of the residue corresponding to 310 of SEQ ID NO: 1.
  • a histidine residue may be included at the position corresponding to residue 310 of SEQ ID NO: 1.
  • the residue corresponding to position 301 of SEQ ID NO: 1 may be one such as leucine.
  • the glycosylation site in the sequence derived from the IgM tailpiece at residue 236 of SEQ ID NO: 1 was disrupted this did not hinder DC-SIGN binding of proteins incorporating this change.
  • polymeric proteins of the invention can be modulated by the insertion of additional glycans, as seen with polymeric proteins of the invention that incorporate the addition of a new glycosylation site (for example at D001 N of SEQ ID NO: 1).
  • mannose-binding receptors such as the mannose receptor (MR)
  • MR mannose receptor
  • oligomannose type glycans found on IgG are associated with enhanced pro-inflammatory effector functions and rapid in vivo clearance (for example by splenic macrophages) of the molecules to which they are attached.
  • polypeptides of the invention in which the glycosylation site corresponding to N77 of SEQ ID NO: 1 is disrupted and/or an additional glycosylation site (such as at residue 1 of SEQ ID NO: 1) is added may have decreased tendency to induce inflammation. They may also have improved half-lives and PK, particularly when used as a biomimetic of IVIG for the treatment of autoimmune disease. Furthermore, the finding that increased valency does not correspond to increased induction of inflammation suggests that embodiments of the proteins of the invention that are fused to other therapeutic molecules (for example for the delivery of a payload of such drugs), may achieve improved yield of a fused therapeutic agent without a concomitant increase in adverse events associated with mannose-dependent receptor-mediated inflammation.
  • the glycans associated with these glycosylation sites in the polymeric proteins of the invention may be modified by selection of the cell type in which the proteins are expressed.
  • Chinese hamster ovary (CHO) cell lines deficient for various glycosylation enzymes are available, and expression of the proteins of the invention in these cell lines is able to yield proteins with a desired glycan composition.
  • terminal sialic acid is involved in DC-SIGN binding, and it is known that growing CHO cells as monolayers (rather than in suspension) is able to enhance sialylation.
  • the proteins may be expressed by CHO cells grown in monolayer culture, and in the case that it is wished to decrease the interaction of polymeric proteins of the invention with DC-SIGN the proteins may be expressed by CHO cells grown in suspension culture.
  • Galactose may bind dectin-1 , and so in the case that it is wished to produce polymeric proteins of the invention with enhanced binding to dectin-1 , these proteins may be expressed in Iec4 mutant CHO cell lines. This expression will be expected to lead to bi- and tri-antennary structures fully capped with galactose. Such proteins may promote the association of FcYRIIb with dectin-1 , thereby blocking C5a-dependent inflammation in vivo. Such proteins of the invention may therefore be of utility in thepreventingon and/or treatment of inflammatory condidtions including peritonitis and skin blistering in epidermolysis bullosa (for example by means of IVIG treatment, which is already known to positively affect these conditions).
  • Polymers and “monomers” The polymeric proteins are composed of multiple polypeptide units referred to as “monomers” or “monomer units” for the purposes of the present disclosure. Each of these monomer units comprises two disulphide bonded chains, each chain being a chimeric protein formed by combining sequence based upon an immunoglobulin G heavy chain and sequence based upon a modified tailpiece region from immunoglobulin M.
  • a "pentamer” will comprise 5 “monomers” for a total of 10 disulphide bonded chimeric protein chains
  • a “hexamer” will comprise 6 “monomers” for a total of 12 disulphide bonded chimeric protein chains
  • a “dodecamer” will comprise 12 “monomers” for a total of 1024 disulphide bonded chimeric protein chains.
  • references to promoting or increasing polymerisation should, except for where the context requires otherwise, be taken as referring to an increase in the size of polymers formed. That is to say, for example, an increase in the proportion of dodecamers formed, as compared to the number of pentamers or hexamers. This increase in the size of polymers formed may optionally occur in combination with an increase in the proportion of the polypeptides being incorporated in polymers.
  • polymeric proteins of the invention may be provided in the form of any suitable multimer, including, but not limited to, pentamers, hexamers, heptamers, nonomers, decamers, undecamers, and dodecamers.
  • a polymeric protein of the invention is in the form of a dodecamer.
  • the C309 residues in neighboring monomers are covalently linked to each other in this model.
  • the tailpiece cysteines in randomly selected monomers are also joined together.
  • FSC forward
  • SSC side
  • A Individual CD19 + B cells stained with anti-human CD19-FITC were gated (middle panel). Binding of 5C ⁇ g of hexa-Fc to gated human CD19 + B lymphocytes was detected using phycoerythrin (PE)-labeled goat (Fab' 2 ) anti-human IgG (right panel).
  • PE phycoerythrin
  • Fab' 2 phycoerythrin
  • Binding of hexa-Fc (right panel) to FcRL5/CD32 double transfectants, FcRL5 single transfectants, FcRL4/CD32 double transfectants and CD32 single transfectants.
  • CD200 transfected controls are omitted from the overlays for clarity. Binding of hexa-Fc and heat-aggregated IgG to the FcRL5/CD32 double transfectants is enhanced when compared with FcRL5 single transfectants.
  • FcRL4/CD32 (top panel) or FcRL5/CD32 (bottom panel) double-transfectants were pre- incubated with anti-FcRL5 blocking mAb 509F6 or anti-FcRL4 blocking mAb 413D12.
  • Anti-FcRL5 blocking antibody did not reduce binding of hexa-Fc but markedly reduced binding of heat-aggregated IgG, showing that hexa-Fc prefers to bind FcyRllb when given the choice of either receptor.
  • Remaining traces show binding by 509F6 and 413D12 in the absence of human Igs.
  • Cell surface expression of FcRL5 and FcRL4 were confirmed using FITC-conjugated anti-FLAG M2 mAb or by staining with anti-CD32 Ab. Data are representative of duplicate experiments.
  • FIG. 1 Binding of hexa-Fc to human DC-SIGN by multi-channel surface plasmon resonance analysis (SPR). Association and dissociation curves of Igs binding to recombinant human DC-SIGN immobilized on a sensor chip. Hexa-Fc, dimeric-Fc or gp120 control were injected at doubling dilutions as indicated into flow at time 0, and replaced with buffer at 300 sec. Data are representative of duplicate experiments.
  • SPR surface plasmon resonance analysis
  • IgM-Fc and PentaglobinTM binding to recombinant human DC-SIGN or SIGNR1 immobilized on a sensor chip were injected at doubling dilutions from 10 ⁇ to 0.32 ⁇ into flow at time 0, and replaced with buffer at 300 sec. Data are representative of duplicate experiments.
  • FIG. 7 (A) /V-glycan profile of hexa-Fc and two different IVIg preparations MALDI-TOF mass spectra of permethylated N-glycans of hexa-Fc, dimeric-Fc, GammaGardTM IVIg, and Malawian IVIg were obtained from the 50% MeCN fraction from a C18 Sep-Pak column ("Materials and Methods").
  • Annotated structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M+Na] + . Putative structures are based on composition, tandem MS/MS, and biosynthetic knowledge.
  • FIG. 8 Microliter wells (Nunc) were coated with DC-SIGN at l O g/m in carbonate buffer pH9 and incubated over night at 4°C prior to blocking for 2b at room temperature (RT) in TSM (20mM Tris-HCI, 150mM NaCI, 2mM CaCI 2 , 2mM MgCI 2 , 5% BSA) buffer pH 7.4. The wells were washed four times with TS before addition of 100 ⁇ digested or undigested antibodies at 1 Qpg/m! in TBS buffer to duplicate wells.
  • TSM room temperature
  • Figure 11 Structure of the glycosylated Fc domain determined from MD simulations Initial structure of the complex. In the bottom view, the atoms of the complex are depicted as van der Waals spheres to more closely reflect the physical structure. The major part of the structure is the hFc, while the the sugars are as depicted in the schematic of the Man 5 GlcNAc 2 glycan shown on the left, where mannose residues are circles, the N- acetylglucosamines are squares, and the asparagine residue is an oval. Two views of the complex, differing by 90° rotation about the long axis, are shown.
  • FIG. 12 Evaluation of the accessibility of the a1 ,6 branch mannose residues for the DC- SIGN CRD.
  • the CRD is positioned so that the mannose residues in the crystal structure overlap those in the Fc-glycan.
  • the van der Waals representation of the structure (right) more closely reflects the physical structure of the complex.
  • N236A and D001 N/N236A mutants run at a molecular weight consistent with the formation of dodecamers
  • N236A mutant binds human monocytes (in particular CD14 low, CD16 high monocytes).
  • Figure 15 illustrates results from a study of DC-SIGN binding by series 1 mutants.
  • Figure 16 illustrates results from a study of activation of complement by proteins of the invention, and comparator proteins.
  • Fc-fusion proteins are a well-established class of therapeutics, in fact presently exhibiting the greatest growth rate of all biologies in the United States. Notwithstanding this success though, there is great interest in identifying novel approaches to improve their efficacy and safety while expanding their range of potential clinical applications to other areas such as vaccines and replacements for intravenous immunoglobulin (IVIG) therapy.
  • IVIG intravenous immunoglobulin
  • one well-recognized drawback of the present Fc-fusion design for many of its potentially new applications is its monomeric structure: it is not able to cross-link multiple receptors with the high affinity required for enhanced function.
  • DC- SIGN intercellular adhesion molecule-3-grabbing non-integrin
  • each of these receptors is also targeted by pathogens in their attempt to inhibit immune responses involved in their removal.
  • FcyRllb, FcRL5, and DC-SIGN may thus limit immune cell activation against chronic pathogens or self-reactive antigen, and approaches that have the potential to target these receptors with high affinity/avidity may prove beneficial in therapies, including IVIG, aimed at controlling proinflammatory disease.
  • hexameric human lgG1-Fc (hexa-Fc) that bound human FcyRI, FcYRIIa R131 , FcyRllb, and mouse FcyRI, FcyRllb with higher affinity than monomeric or dimeric human lgG1-Fc, as expected from its increased valency (Czajkowsky et al, 2012; Mekhaiel et al, 201 1) (Fig 1).
  • hexa-Fc binds to human circulating B cells and monocytes.
  • CD19 + B cells from peripheral blood mononuclear cells of healthy human volunteers were screened by flow cytometry analysis.
  • the anti-human IgG detecting reagent most likely due to direct interactions with the IgG B cell receptor (BCR) and/or pre-bound IgG found on B cells, we nonetheless detected increased binding of hexa-Fc (Fig 2A).
  • BCR IgG B cell receptor
  • Fig 2A We could also observe a robust association of hexa-Fc to CD14 +
  • FcRL5 and Fc ⁇ Rllb are receptors for hexameric IgG1-Fc
  • Hexa-Fc was previously shown not to bind human FcRn (Mekhaiel et al, 201 1 ).
  • the binding site for FcRn on IgG is localized within the CY2-CY3 junction and involves residues Ile253, His310, His433 and His435 (Rath et al, 2013; Shields et al, 2001 ).
  • the pKa of histidine is 6.0-6.5 such that several histidine residues become protonated below physiological pH, allowing for salt bridge formation with acidic residues on the FcRn, thus explaining the strict pH dependency of IgG-FcRn interactions (Raghavan et al, 1995).
  • Hexa-Fc binds DC-SIGN in a valence dependent manner
  • overall affinity was lower when compared with gp120. This is to be expected from the lower density of favoured high-mannose glycans on the Fc polypeptide.
  • IgM-Fc and IVIg enriched for polymeric Igs also bind DC-SIGN
  • Pentaglobin ® a clinically available IVIg preparation used in the treatment of sepsis and enriched for polymeric Igs (12% IgM, 12% IgA and 76% IgG by weight) (Hoffman et al, 2008).
  • Pentaglobin ® bound human DC-SIGN but not SIGNR1 (Fig 6), a finding that may be attributed to differences in glycans or other undetermined posttranslational modifications that arise when expressing proteins in CHO cells.
  • MS analysis of hexa-Fc revealed a paucity of sialylated structures but enrichment for high mannose glycans (Man 5 GlcNAc 2 , Man 6 GlcNAc 2 ). This glycan profile is also consistent with observations that DC-SIGN binds high mannose structures (van Liempt et al, 2006). MS/MS fragmentation was performed on ions whose masses were consistent with the presence of fucose in order to determine whether hexa-Fc contains terminal antennal fucose residues such as in the Lewis X antigen which can also bind DC-SIGN. These experiments ruled out antennal- linked fucose.
  • MS/MS of m/z 2244 shows a core rather than terminal location for the fucose (Fig. 7B), indicating that the DC-SIGN binding affinity for hexa-Fc is likely the result of increased avidity binding mediated by mannose.
  • the MS analysis also revealed hexa-Fc to be richer in larger multi-antennary and polylac containing N-glycans (for example m/z 2693, 3143 and 3504) which would present more terminal galactose when compared to IVIg N-glycans (Fig 7).
  • Hexa-Fc binds complement C1q and activates complement via the classical pathway
  • FcRL5 interacted with both Fc heavy chains, one predominantly in the D1/D2 junction and the other within the D2 domain, although the number of these associations were significantly lower than in the FcyRIII/Fc complex.
  • the heavy chain interaction with the D1/D2 junction in FcRL5 was markedly weaker than in FcyRIII complex.
  • both glycan chains essentially adopt one of two configurations: one in which the di-N- acetylchitobiose core, the central ⁇ mannose, and a1-6 branch residues are all in close proximity to the C Y 2 domain (similar to the glycan structures observed in earlier crystallographic studies (Crispin et al, 2009; Harris et al, 1997; Matsumiya et al, 2007)) and a previously uncharacterized configuration in which only the di-N-acetylchitobiose core is close to the C Y 2 domain.
  • the a1-3 branch mannose residue in both configurations is essentially always oriented towards and frequently interacting with the other glycan chain.
  • FcRL5 might be a receptor for IgG (Ehrhardt et al, 2003; Haga et al, 2007). However, binding of soluble monomeric IgG to FcRL5-transfected 293 cells was not observed in FACS-based assays, indicating that FcRL5 was likely to be a low- to medium-affinity Fc receptor, if at all (Wilson et al, 2012).
  • DC-SIGN signalling invokes IL-10 production which is of significance in anti-inflammatory pathways (Gringhuis et al, 2009; Gringhuis et al, 2007).
  • DC- SIGN and SIGNR1 are important receptors in the efficacy of IVIg in controlling autoimmune disease (Anthony et al, 2011 ; Schwab et al, 2012) prompted us to investigate the interaction of these additional receptors with hexa-Fc (Fig 5).
  • IgM-Fc or IgM-enriched IVIg can also bind DC-SIGN (Fig. 6).
  • Human IgM is known to be heavily mannosylated and these glycans are involved in binding of IgM by mannan binding lectin, a member of the collectin family of proteins, which bind to oligomannose and GlcNAc- terminating structures (Arnold et al, 2005).
  • hexa-Fc may be superior to IVIg at promoting the association of FcuRllb with dectin-1 , thereby blocking C5a-dependent inflammation in vivo, including peritonitis and skin blistering in experimental epidermolysis bullosa acquisita (EBA) for which IVIg is known effective (Czernik et al, 2012).
  • the multivalent state of the Fc is fundamental to designing reagents that bind inhibitory Fc- and/or glycan receptors optimally (Karsten et al, 2012).
  • presenting glycan residues on the multimeric cylindrical-Fc barrel or bird-cage structure would likewise be expected to significantly enhance receptor binding as we have shown here for hexa-Fc interactions with Fc Rllb, FcRL5, and DGSIGN and thus may have greater potential for inhibiting autoimmune disease when translated to an IVIg biomimetic therapy (previous PCT as reference for support of structure).
  • Hexa-Fc also binds C1 q and leads to C5b-9 deposition when coated down onto ELISA plates (see Fig 9).
  • a fusion partner greatly interferes with the ability of hexa-Fc to engage complement and FcyRs (Mekhaiel et al, 2011).
  • the lack of binding to FcyRs and C1q is due to the fusion partner blocking access to the FcyR and C1q binding sites (the amino-terminal region of C 2 domain, also the lower hinge region) or to a lack of receptor flexibility when fused in the existing hinge architecture (Oi et al, 1984; Saphire et al, 2002; Sondermann et al, 2000).
  • the hinge region serves as a spacer and mediates segmental flexibility allowing the fusion partner to assume a variety of orientations in space relative to the Fc (Saphire et al, 2002). Modifications to the existing hinge e.g. use of the extended hinge from human lgG3, may therefore move the fusion partner away from the critical FcyRs and C1 q binding sites and thereby reinstate effector functions to hexameric Fc-fusions that are critical for clearance.
  • IVIg The inhibitory properties of IVIg are known to result from its ability to scavenge active complement proteins, and studies have shown that high levels of IgG can inhibit the uptake of C3b and C4b onto the surface of sensitized guinea pig and human erythrocytes and prevent complement-mediated tissue damage (Basta et al, 1989; Basta et al, 2003). It is thought that IVIg binds the activated complement components C3b and C4b and therefore prevents their deposition onto target surfaces.
  • a disease model in which IVIg convincingly modulates complement is dermatomyositis (Orange et al, 2006).
  • Dermatomyositis is an inflammatory myopathy mediated by the deposition of complement and formation of the C5-9 membrane attack complex (MAC) on intramuscular capillaries, leading to loss of capillaries and muscle ischemia and necrosis.
  • MAC C5-9 membrane attack complex
  • hexa-Fc may therefore be a useful therapy for dermatomyositis.
  • mutations that disrupt C1q binding e.g. K322A, P329A, P331A may be introduced into the wild-type molecules described herein.
  • a critical feature to the utility of oligomeric Fc-fusion proteins in future drugs or vaccines will be their ability to interact with the FcRn.
  • His310 is critical to binding of hexa-Fc to human FcRn, although whether this reinstates binding in the context of N-terminal fusions remains to be tested.
  • the nature of the fused partner may potentially affect FcRn binding and interactions with FcRn will therefore most likely need to be determined for each unique fusion.
  • Hexa-Fc now provides a template molecule to further engineer selective gain-of and/or loss-of function mutations, as demonstrated here for FcRn, that allow the exisiting multimeric scaffold to be tailored for optimal use in novel drugs and vaccines.
  • CL309/310CH mutant was constructed by PCR overlap extension mutagenesis from the wild-type vector (pFUSE-hlgG1-Fc-TP-LH309/310CL) as the template, using the internal mismatched primer mut-3:5'-ACCGTCTGCCACCAGGACTGG-3' and its complement to incorporate a CTC to CAC substitution and Fcmut-1 :5'- ACCCTGCTTGCTCAACTCT-3' and Fcmut-1 :3'-TTGATGAGTTTGGACAAACCA-5' as flanking primers.
  • PCR products were then digested using EcoR ⁇ and Nhe ⁇ (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hlgG1-Fc-TP- CL309/310CH.
  • EcoR ⁇ and Nhe ⁇ New England Biolabs
  • the entire coding sequence of the new expression plasmid was sequenced on both strands.
  • CHO-K1 cells European Collection of Cell Cultures
  • FuGene Promega
  • plasmid plasmid using FuGene (Promega)
  • Positive clones selected.
  • Cells were grown in DMEM complete media supplemented with 10% ultra-low bovine IgG FCS, 100 lU/ml penicillin, and 100 ⁇ gml -1 streptomycin (PAA) at 37°C/5%C02.
  • Stable transfectants were selected in medium containing 400 ⁇ gml -1 of Zeocin (Invivogen).
  • Clones secreting hexa-Fc proteins were detected by sandwich enzyme-linked immunosorbent assay (ELISA) using goat anti- human IgG (Invitrogen) to capture and goat anti-human IgG-Fc (Sigma-Aldrich: A0170) conjugated to horseradish peroxidase to detect
  • ELISA sandwich enzyme-linked immunosorbent assay
  • goat anti-human IgG Invitrogen
  • goat anti-human IgG-Fc Sigma-Aldrich: A0170 conjugated to horseradish peroxidase to detect
  • the CL309/310CH mutant was purified from larger scale cultures on Protein-G-Sepharose (GE Healthcare, Little Chalfont, Bucks, UK) using an AKTA FPLC (GE Healthcare).
  • PBMC peripheral blood mononuclear cells
  • cDNA encoding human CD200R, FcRL4, or FcRL5 were ligated into pFLAG-CMV-3 (Sigma).
  • cDNA encoding human CD32 was ligated into pEF6 (Invitrogen) (Wilson et al, 2012). Proteins were expressed in 293 cells by transient transfection using Lipofectamine 2000 (Wilson et al, 2012). Transiently transfected 293 cells were used for Ig binding assays 36-42 h after transfection.
  • Purified human lgG1 was obtained from Sigma-Aldrich and hexa-Fc was purified as previously described (Mekhaiel et al, 201 1).
  • Igs were aggregated by heating to 60°C for 30 min. Igs were then diluted to 100 ⁇ g/ml in PBS/1 % BSA. The 293 cells were incubated for 30 min on ice with the Igs and washed four times, followed by incubation with biotin-conjugated goat F(ab') 2 anti-human IgG (Southern Biotechnology) for 20 min on ice.
  • Recombinant human DC-SIGN was generated as described previously (Mitchell et al, 2001). Recombinant SIGNR1 was from R and D systems. Purified recombinant HIV gp120 was a kind gift of Dr Max Crispin (University of Oxford).
  • Soluble recombinant DC- SIGN and SIGNR1 proteins were captured on GLM sensor chips (Bio-Rad laboratories) via amine coupling with sulfo-N-hydroxysuccinimide/1-Ethyl-3-[3- dimethylaminopropyl]carbodiimide and all sensorgrams using soluble-phase analytes of immunoglobulin preparations were recorded at 25°C with the ProteOn XPR36 surface plasmon resonance biosensor (Bio-Rad laboratories) at a flow rate of 25 ⁇ I per minute. Kinetic parameters for protein-protein interactions were determined using the 1 : 1 Langmuir modeling algorithms included in the ProteOn Manager software suite (Bio-Rad Laboratories).
  • Microtiter wells (Nunc) were coated with titrated amounts of the Fc-fusions (20.0- 0.1 ⁇ g/ml) in PBS and incubated over night at 4°C prior to blocking with 4% skimmed milk (Acumedia) for 1 h at room temperature (RT).
  • the wells were washed four times with PBS/0.005% Tween 20 (PBS/T) pH 6.0 before addition of GST-tagged hFcRn in 4% skimmed milk PBS/T pH 6.0 and added to the wells (Andersen et al, 2008).
  • N-glycomic analysis was performed according to a protocol described previously (North et al, 2010). Briefly, 50 ⁇ g of each sample was reduced by dithiothreitol (Sigma, Aldrich) and then carboxymethylated by iodoacetic acid (Sigma Aldrich). Samples were subsequently dialyzed, freeze-dried and digested by trypsin (Sigma Aldrich). The peptides/glycopeptides were purified using Oasis HLB Plus Short cartridges (Waters). N- glycans were released from glycopeptides by PNGaseF (Roche Applied Science) and isolated from peptides using Sep-Pak C18 catridges (Waters).
  • the released N-glycans were permethylated, purified by Sep-Pak C18 cartridges again, freeze-dried and dissolved in 10D I methanol.
  • 1 zi I of dissolved sample was premixed with (III of matrix (for MS, 20 mg/ml 2,5-dihydroxybenzoic acid in 70% (v/v) aqueous methanol; for MS/MS, 20 mg/ml 3,4-diaminobenzophenone in 75% (v/v) aqueous acetonitrile).
  • MALDI-TOF MS analysis using a Voyager-DETM STR mass spectrometer (Applied Biosystems). The data were analyzed using Data Explorer (Applied Biosystems) and Glycoworkbench (Ceroni et al, 2008).
  • the homology model of FcRL5 was constructed with the automated homology modeling tools in DeepView (Guex & Peitsch, 1997), using the human FcRL5 (accession no. Q96RD9) and the crystal structure of the FcyRI (PDB accession codes: 3RJD). The structure (and all models here) was then solvated in TIP3 water (Jorgensen et al, 1983) and then minimized and equilibrated using VMD/NAMD (Phillips et al, 2005) and the CHARMM36 force field (Best et al, 2012), in the constant pressure and constant temperature (NPT, 295K, 1atm) ensemble.
  • the temperature and pressure were controlled by the Berendsen thermostat and barostate with a coupling time of 0.1 ps and 1.0ps, respectively.
  • the particle mesh Ewald algorithm was employed to treat electrostatic interactions.
  • the van der Waals interactions were treated with a cut-off of 12A, and the integration step was set to 2fs. After ⁇ 10ns, the protein attained an equilibrated conformation, as judged by the root-mean-square deviation of the protein backbone.
  • the protein secondary and tertiary structures were evaluated with VMD.
  • the Fc domain used in these simulations was the human Fc structure of the FcyRIII/Fc complex.
  • the crystal structure of the human lgG1 (PDB accession codes: 2WAH) was used as the template and initial structure for the model, as it contained high mannose glycans.
  • the glycans present in this structure (Man 9 GlcNAc 2 ) were not found to be attached to hexa-Fc by MS, we manually removed the appropriate mannose residues to obtain the initial structure of the Man 5 GlcNAc 2 glycan, which is attached to the hexa-Fc as shown here by MS (circled in Fig 7).
  • hexa-Fc has been previously described in the inventors' earlier patent applications.
  • Each mutant was constructed by PCR overlap extension mutagenesis from the wild-type vector (pFUSE-hlgG1-Fc-TP-LH309/310CL) as the template, using the internal mismatched primers described in Table 2 below and Fcmut1-F: ACCCTGCTTGCTCAACTCT and Fcmut1-R: TG GTTTGTC C AA ACTC ATC AA as flanking primers.
  • Fcmut1-F ACCCTGCTTGCTCAACTCT
  • Fcmut1-R TG GTTTGTC C AA ACTC ATC AA as flanking primers.
  • N007A was used as the template for N236A, giving N077A/N236A.
  • the same approach was used to create the D001 N knockouts on the D001 template.
  • PCR products were then digested using EcoR ⁇ and Nhe ⁇ (New England Biolabs) and cloned back into the wild-type vector to generate each mutant.
  • EcoR ⁇ and Nhe ⁇ New England Biolabs
  • the entire coding sequence of the new expression plasmid was obtained on both strands.
  • CHO-K1 cells European Collection of Cell Cultures
  • FuGene Promega
  • NXS/T N-linked glycosylation site An important feature of hexa-Fc functionality is the NXS/T N-linked glycosylation site, which gives the recombinant protein an almost mandatory glycosylation of the asparagine residues from the NXS/T site. Glycosylation is important in increasing solubility and in influencing interactions with both glycan and Fc-receptors.
  • Hexa-Fc contains two N-linked glycan attachment sites found at Asn77 (equivalent to Asn297 in the Fc of lgG1) and Asn236 in the tailpiece (equivalent to Asn563 of the IgM tailpiece).
  • these carbohydrates were removed from hexa-Fc with peptide N- glycosidase (PNGase) F, and their resulting ability to bind human DC-SIGN investigated by ELISA (Fig. 8).
  • hexa-Fc therefore imposes significant alterations to both the composition and spacio-functional structure of glycans attached to Asn77 and Asn236 that could not have been predicted from prior art based on the glycan compositions of native IgG, or monomeric Fes (see Example 1).
  • These unique glycan structures in hexa-Fc will by definition alter the function of the molecule in novel and unique ways.
  • CHO cell variants as in lec series of CHO cells
  • Fc-fusions as antigen delivery vehicles where you do not wish to drive tolerance or inhibitory responses via DC-SIGN, FcRL5 or FcgRllb.
  • mutants have been generated on the hexa-Fc template (SEQ ID NO: 1) and show unexpected properties that could not have been anticipated from the prior art (discussed above and below).
  • SEQ ID NO: 1 The same panel of glycan mutants are also being introduced into the Fc of lgG2, lgG3 and lgG4.
  • hexa-Fc 2.87 ⁇ g/ml
  • D001 N 9.2 g/ml
  • N77A 0.87 g/ml
  • N236A 1.57 g/ml
  • N77A/N236A 0.72 g/ml
  • D001 N/N236A 0.63 g/ml
  • D001 N/N77A 0.25 g/ml
  • D001 N/N77A/N236A 2.89 ⁇ g/ml.
  • the D001 N variant binds more poorly to DC-SIGN or other glycan receptors.
  • N236A SEQ ID NO:3
  • D001/N236A SEQ ID NO:7 in which the glycan residue in the carboxy-terminal tailpiece were removed run with molecular weights approaching -650 kDa and -700 kDa respectively (red arrow in Fig. 16 and confirmed by SEC).
  • the N236A mutant (SEQ ID NO:3) bound strongly to human monocytes (in particular CD14 low, CD16 high) and to CD19+ B cells (Fig. 14), and the removal of this glycan did not adversely affect binding to DC-SIGN.
  • dodecameric IgM, dodecameric IgGs, and/or IgM- Fc is likely not possible given additional constraints imposed by the size of the Fc (extra Cy2 domain in the Fc) and associated F(ab) 2 arms in each monomer of Ig.
  • IgM differs significantly from hexa-Fc not only with respect to the number and type of N-linked glycans in the Fc but also in that the Cy2 domain replaces the hinge region. Therefore mutations derived from work on IgM or IgM-Fc don't necessarily translate to either structural or functional equivalence in the N236A, D001/N236A mutants (SEQ ID NO:3 and SEQ ID NO:7 respectively) or hexa-Fc.
  • Hexa-Fc has already been shown to be effective at treating idiopathic thrombocytopenic purpura (ITP) in mice. No obvious adverse reactions are observed in these studies when this polymeric protein is administered by the i.p. route.
  • the inventors are further testing the properties of hexa-Fc and proteins of the invention in models of dermatomyositis, bullous pemphigoid, and chronic inflammatory demyelinating polyneuropathy in the expectation that they will prove therapeutically useful in the treatment of these conditions.
  • proteins of the invention have a propensity to polymerise to a greater extent than would be expected from the prior art. Indeed, certain proteins (particularly those comprising SEQ ID NO:3 and SEQ ID NO:7) allow formation of dodecamers with enhanced glycan attachments at the N-terminus that may allow for improved binding to DC-SIGN when expressed as dodecamers rather than hexamers.

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Abstract

L'invention concerne des protéines polymères qui comprennent deux ou davantage de motifs monomères polypeptidiques, chaque motif monomère comprenant deux chaînes de protéines chimères. Chaque motif monomère polypeptidique chimère comprend une partie de liaison de récepteur Fc comprenant deux régions constantes de chaîne lourde d'immunoglobuline G, chaque région constante de chaîne lourde d'immunoglobuline G comprenant un résidu cystéine qui est lié par l'intermédiaire d'une liaison disulfure à un résidu cystéine d'une région constante de chaîne lourde d'immunoglobuline G d'un motif monomère polypeptidique adjacent. Chaque chaîne de protéines chimères comprend également une région de queue d'immunoglobuline M modifiée, la séquence d'acides aminés de chaque monomère polypeptidique chimère comprenant une altération de la structure primaire par rapport aux séquences natives à partir desquelles la région constante de chaîne lourde d'immunoglobuline G ou la région de queue d'immunoglobuline M sont dérivées, ladite altération modifiant le nombre de sites de glycosylation d'une manière qui active la polymérisation. Cette activation de la polymérisation peut conduire à la génération de formes tétramères, hexamères voire dodécamères des protéines. Ces protéines conviennent pour des utilisations médicales, par exemple pour prévenir ou traiter des maladies auto-immunes telles que la thrombocytopénie auto-immune. L'invention concerne également des méthodes de traitement utilisant ces protéines polymères et des compositions pharmaceutiques comprenant lesdites protéines polymères.
PCT/GB2015/052098 2014-07-18 2015-07-20 Proteines polymeres et utilisations de celles-ci Ceased WO2016009232A1 (fr)

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US20210024615A1 (en) * 2018-03-16 2021-01-28 Liverpool School Of Tropical Medicine Chimeric fc receptor binding proteins and uses thereof
US11034775B2 (en) 2016-06-07 2021-06-15 Gliknik Inc. Cysteine-optimized stradomers
US11331372B2 (en) 2016-12-09 2022-05-17 Gliknik Inc. Methods of treating inflammatory disorders with multivalent Fc compounds
CN114621350A (zh) * 2021-06-17 2022-06-14 东莞市朋志生物科技有限公司 嵌合的免疫球蛋白
US12122836B2 (en) 2015-07-24 2024-10-22 Gliknik Inc. Fusion proteins of human protein fragments to create orderly multimerized immunoglobulin Fc compositions with enhanced complement binding

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12122836B2 (en) 2015-07-24 2024-10-22 Gliknik Inc. Fusion proteins of human protein fragments to create orderly multimerized immunoglobulin Fc compositions with enhanced complement binding
US11034775B2 (en) 2016-06-07 2021-06-15 Gliknik Inc. Cysteine-optimized stradomers
WO2018018047A2 (fr) 2016-07-22 2018-01-25 Gliknik Inc. Protéines de fusion de fragments de protéines humaines utilisées afin de créer des compositions de fc d'immunoglobuline multimérisée de manière ordonnée avec une liaison de récepteur fc améliorée
US11331372B2 (en) 2016-12-09 2022-05-17 Gliknik Inc. Methods of treating inflammatory disorders with multivalent Fc compounds
US12337026B2 (en) 2016-12-09 2025-06-24 Gliknik Inc. Methods of treating inflammatory disorders with multivalent Fc compounds
US20210024615A1 (en) * 2018-03-16 2021-01-28 Liverpool School Of Tropical Medicine Chimeric fc receptor binding proteins and uses thereof
AU2019235513B2 (en) * 2018-03-16 2026-01-29 Liverpool School Of Tropical Medicine Chimeric Fc receptor binding proteins and uses thereof
CN114621350A (zh) * 2021-06-17 2022-06-14 东莞市朋志生物科技有限公司 嵌合的免疫球蛋白

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