IMMUNOMODULATORY PROTEINS
FIELD OF THE INVENTION
The invention relates to engineered proteins having an immunomodulatory function, and
their medical uses for treatment of autoimmune or inflammatory diseases. In particular,
the engineered proteins may be used as replacements for intravenous immunoglobulin
(IVIG).
BACKGROUND OF THE INVENTION
Autoimmune and inflammatory diseases are responsible for substantial morbidity and
mortality. In 2003, autoimmune diseases were the sixth most frequent underlying cause
of death in all age groups below 75 years. One important treatment modality is
intravenous immunoglobulin (IVIG) or IgG. Immune globulin products from human
plasma were first developed to treat immune deficiencies. However, seventy percent of
prescribed IVIG is now used for the treatment of autoimmune or inflammatory conditions.
The worldwide consumption of IVIG increased from 300kg per year in 1980 to 100
tonnes per year in 2010. IVIG is derived from the pooled plasma of ~3,000 anonymous
donors, according to a lengthy and expensive manufacturing process. The need for
extensive screening of donors and donated plasma for viruses contributes to the high
cost. Given the increasing demand, and the strict regulation of IVIG production,
shortages of IVIG can occur. IVIG preparations may be subject to inadequate sterility,
the presence of impurities and lot-to-lot variation. They may vary greatly in their
immunoglobulin A content, and IgA can cause allergic or anaphylactic reactions in IgAdeficient
recipients, making them unsuitable for some patients. Very large doses of IVIG
have to be given to patients, typically 2 g per kg bodyweight, and this can cause adverse
reactions in some patients. In view of these limitations, defined replacements for IVIG
are needed.
Although the mechanisms of action for IVIG remain unclear, Fc-fragments derived from
IVIG can cure children suffering from idiopathic thrombotic purpura (ITP) (Debre M et al,
1993). Interactions between the Fc portion of IgG with both inhibitory and activating
FcyRs found on monocytes and macrophages are considered to be important, although
the exact receptors involved are hotly debated (Samuelsson A. et al, 2001; Bazin R. et
al, 2006; Crow. A.R. er al, 2003; Leontyev D. et al, 2012; Siragam. V. et al, 2006) and
may vary for the disease for which IVIG is used (Araujo L.M. er al, 201 1; Anthony R.M. et
al, 201 1). The inhibitory Fc receptor FcyRllb has been shown to be necessary for the
protective effect of IVIG in a mouse model of ITP (Samuelsson et al, 2001). A role of the
activating Fc receptor FcyRllla has also been demonstrated in various diseases
(reviewed Mekhaiel et al, 201 1b). The FcRn responsible for maintaining the long-half-life
of the Fc in plasma has also been postulated to be involved (Roopenian & Akilesh,
2007), although recent work has shown that it is not involved in ameliorating ITP in the
mouse model (Crow et al, 201 1). Fc portions interact not only with Fc receptors, but also
certain lectins. It is known that IVIG binds to CD22 lectin (Siglec-2) on B lymphocytes via
a terminal sialic acid on the Fc-glycan (Seite J.F. et al, 2010), and a recent study
demonstrated that sialylated Fes are responsible for the anti-inflammatory effects of IVIG
in a mouse model of arthritis as a result of their interaction with human lectin receptor,
DC-SIGN, on DCs (Anthony R.M et al, 201 1).
IVIG may also work via a multi-step model where the injected IVIg first forms a type of
immune-complex in the patient (Clynes et al, 2005; Siragam et al, 2005, 2006; Machino
et al, 2012). Once these immune-complexes are formed, they interact with these
receptors to mediate anti-inflammatory effects helping to reduce the severity of
autoimmune disease or the inflammatory state (Siragam et al, 2006). Multimers of Fc
are formed in IVIG by anti-idiotype interactions (Machino et al, 2010; Machino et al,
2012; Roux & Tankersley, 1990; Teeling et al, 2001) or by covalent interactions of the Fc
(Yoo et al, 2003). IgG multimers are postulated to show higher avidity binding to the
above receptors and by nature of cross-linking the said receptors induce protective
signals that are not induced by monomers of Fc. This is supported by the reversal of ITP
in mice by immune-complexes (ICs) (Bazin et al, 2006), and by the observation that ICs
enhance tolerogenicity of immature dendritic cells via FcyRllb to promote attenuation of
lupus (Zhang et al, 201 1). The proportion of multimeric IgG and/or sialylated IgG in
commercially available IVIG is extremely low (< 1% and 5% respectively), which may
contribute to the need to administer large quantities (Nimmerjahn & Ravetch, 2007).
Other proposed mechanisms of action of IVIG are restoration of the idiotypic—antiidiotypic
network; suppression or neutralization of cytokines by specific antibodies in the
IVIG; blockage of binding of adhesion molecules on leukocytes to vascular endothelium;
Inhibition of complement uptake on target tissues; neutralization of microbial toxins;
blockage of Fas ligand-mediated apoptosis by anti-Fas antibodies in the IVIG; induction
of apoptosis with anti-Fas antibodies at high concentrations of IVIG; neutrophil apoptosis
by anti-Siglec-9 antibodies in IVIG; saturation of the FcRn receptors to enhance the
clearance of autoantibodies; induction of inhibitory FcyRllb receptors on effector
macrophages; neutralization of growth factors for B cells, such as B-cell activating factor;
inhibition of T cell-proliferative responses; expansion, activation, or both of a population
of Treg cells; inhibition of the differentiation and maturation of dendritic cells;
enhancement of the differentiation and maturation of "primed" dendritic cells (reviewed in
Ballow, 201 1; Mekhaiel et al, 201 b).
Recombinant proteins for use in treating autoimmune diseases and/or as IVIG
replacement compounds have been proposed. US 201 1/0081345 (Moore) discloses
single chain Fc (scFc) proteins, having one Fc unit per molecule, composed from two
linked Fc domain amino acid chains, which may be useful for treating autoimmune
disease. US 2004/0062763 (Temple University; Mosser) discloses use of multivalent
Abs or portions thereof to ligate FcyRI to upregulate IL-10 production, to treat
autoimmune disorders. The agent may be two or more Fc fragments coupled together or
provided in a single recombinant peptide. US 2008/0206246 (The Rockefeller University;
Ravetch) discloses a polypeptide containing at least one IgG Fc region, which is
sialylated, and its use as IVIG replacement compound. Although the compounds
disclosed in these documents might target Fc receptors, monomeric or dimeric Fccontaining
compounds may not bind to Fc receptors with sufficient avidity to be effective
or fully effective as IVIG replacement compounds. They would therefore not be suitable
biomimetics of the multimeric fraction of IVIG. Higher order multimers are not disclosed
in these documents. Neither is any means of engineering higher order multimers which
are active as IVIG replacement compounds.
US 2010/0239633 (University of Maryland, Baltimore; Strome) discloses IVIG
replacement compounds comprising multiple linked Fc portions. Star-shaped
arrangements are envisaged using the m4 domain of IgM and the J chain to effect
polymerisation. However, working examples were not described, and computer
simulations reported herein suggest that exemplary molecules would not polymerise
effectively and/or that the Fc portions would not be arranged for effective binding of Fc or
other receptors. In addition, complex biologicals containing more than one different
polypeptide chain may be more difficult to manufacture to uniformity, because not all of
the polypeptide subunits may interact in a stable and predictable way. For example, cell
lines expressing antibody heavy and light chains which produce active intact antibody
containing the correct proportion of light and heavy chains may also produce
therapeutically inactive heavy chain dimers without light chain attachment, or halfmer
molecules.
Linear structures were also proposed in US 2010/0239633, in which individual amino
acid chains dimerize by pairing between identical Fc domain amino acid chains, thus
generating Fc regions. For example, hinge regions may form inter-chain disulphide
bonds between two individual amino acid chains. However, where multiple Fc amino
acid chains are linked in series, there may be multiple different ways in which these
chains may pair, as illustrated in Figures 11 and 12 of US 2010/0239633. Thus, a
uniform product with reliable and predictable properties would not be expected. It is
proposed to include a terminal heavy chain domain from IgE to prevent this "zippering"
effect. However, binding of the molecules to IgE receptors could have unwanted
consequences, including the risk of anaphylactic shock or other allergic responses.
US 2010/0239633 also suggests making cluster molecules in which multimerizing
regions, such as lgG2a hinge or isoleucine zippers, cause amino acid chains to dimerize
or multimerize, thus bringing together pairs of Fc domain amino acid chains to form
functional Fc units. In a follow on study, Jain et al (2012) describe the production of
recombinant proteins in which the human lgG2 hinge sequence or the isoleucine zipper
was fused to murine lgG2a. The authors report that either multimerizing region caused
the formation of homodimers (i.e. single functional Fc units referred to herein as
monomers) and multimers. Multimeric fractions of these proteins had some efficacy in
treatment of a mouse model of ITP or in the collagen-induced arthritis mouse model.
However, the bulk of the produced protein containing the lgG2 hinge was monomeric, or
existed as dimers or trimers. Higher order oligomers accounted for only a small
proportion, with unknown quaternary structure. In proteins containing the isoleucine
zipper, the proportion of higher order multimers was lower. The higher order multimers
appeared heterogeneous in size, and their structures, including the nature of the glycans
attached at N297, are unknown. Isolating some or all of these multimers for therapeutic
use would necessitate substantial waste given their low proportion in the produced
protein, and appears unlikely to be commercially viable.
A need remains for compounds of defined structure which effectively target appropriate
mechanisms underlying the biological activity of IVIG, for treating autoimmune and
inflammatory diseases.
The listing or discussion of a prior-published document in this specification should not be
taken as an acknowledgement that the document is part of the state of the art or is
common general knowledge.
SUMMARY OF THE INVENTION
A first aspect of the invention provides a method for treatment of a mammalian subject
for an autoimmune or inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
comprising five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion
comprising two immunoglobulin G heavy chain constant regions;
wherein 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;
wherein the polymeric protein does not comprise a further immunomodulatory portion; or
an antigen portion that causes antigen-specific immunosuppression when administered
to the mammalian subject.
A second aspect of the invention provides a method for treatment of a mammalian
subject for an autoimmune or inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
consisting of five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion
consisting of two immunoglobulin G heavy chain constant regions; and, optionally, a
polypeptide linker linking the two immunoglobulin G heavy chain constant regions as a
single chain Fc; and
wherein 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.
A third aspect of the invention provides a method for treatment of a mammalian subject
for an autoimmune or inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
consisting of five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion and a
tailpiece region;
wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain
constant regions; and, optionally, a polypeptide linker linking the two immunoglobulin G
heavy chain constant regions as a single chain Fc;
wherein each modified human immunoglobulin G heavy chain constant region comprises
a cysteine residue which is linked via a disulfide bond to a cysteine residue of a modified
human immunoglobulin G heavy chain constant region of an adjacent polypeptide
monomer unit; and
wherein the tailpiece region is fused to each of the two modified human immunoglobulin
G heavy chain constant regions of the polypeptide monomer unit, and facilitates the
assembly of the monomer units into a polymer.
A fourth aspect of the invention provides a polymeric protein comprising five, six or seven
polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion
comprising two immunoglobulin G heavy chain constant regions;
wherein 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;
wherein the polymeric protein does not comprise a further immunomodulatory portion; or
an antigen portion that causes antigen-specific immunosuppression when administered
to a mammalian subject;
wherein each polypeptide monomer unit does not comprise a tailpiece region fused to
each of the two immunoglobulin G heavy chain constant regions.
A fifth aspect of the invention provides a polymeric protein consisting of five, six or seven
polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion
consisting of two immunoglobulin G heavy chain constant regions; and, optionally, a
polypeptide linker linking the two immunoglobulin G heavy chain constant regions as a
single chain Fc; and
wherein 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.
A sixth aspect of the invention provides a nucleic acid molecule comprising a coding
portion encoding a polypeptide monomer unit of a polymeric protein as defined according
to the fourth or fifth aspect of the invention.
Further aspects of the invention are an expression vector comprising the nucleic acid
molecule of the sixth aspect of the invention; a host cell comprising the expression
vector; and a therapeutic composition comprising the polymeric protein of the fourth or
fifth aspect of the invention.
Medical uses corresponding to the methods of treatment of the first to third aspects of
the invention are also envisaged.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig 1: Hexameric-Fc and structural characterization. A Model of hexameric Fc
showing Cys309 and Cys360 (tailpiece) disulphide linkages formed between one
monomer and its adjacent monomers (left panel). Tapping mode atomic force
microscopy images (right hand panels) reveal barrel shaped, six-fold symmetric
complexes consistent with hexamers. At smaller scan sizes (lower of the two right hand
panels), these complexes are shown to be ~20nm in diameter (scale bar in lower panel
is 50nm). B The hexameric-Fc produced from mammalian cell lines can be detected as
molecules of approximately 312 kDa by size-exclusion chromatography (trace
highlighted as hexamer). A small proportion of dimers is also detected.
Fig 2: Binding of hexameric-Fc to human and mouse Fcy-receptors (FCYRS).
Titrated amounts (50-0.3 nM of Fc proteins were coated onto ELISA wells. Binding by
human or mouse glutathione-S-transferase (GST)-fused FcyRs as indicated were
visualized using horse-radish peroxidase (HRP)-conjugated anti-GST antibodies. Values
represent triplicate determinations. Error bars are standard errors (SE) around the mean.
Antigen-fused Fc proteins bound poorly to Fc receptors, as described in Mekhaiel et al,
201 1a.
Fig 3: Binding of hexameric-Fc to human CD19+ human B lymphocytes.
Characteristic flow cytometry plot showing different populations of human leucocytes
represented by their forward and side-scatter profiles (left panel). Individual CD19+ B
lymphocytes stained with anti-human CD19-FITC (boxed, middle panel) were gated and
investigated for binding of hexameric-Fc (right panel). Binding of 50 g of hexa-Fc to
CD19+ B cells is indicated by the furthest trace on the right (arrowed hexa-Fc). Prior
incubation of cells with monoclonal antibody 509F6 specific for FcRL5 (arrowed hexa-Fc
+ FcRL5 blocking mAb 509F6) ablated binding of hexameric-Fc indicating that FcRL5
was responsible for some of the binding of hexameric-Fc to B cells.
Fig 4: Complement binding analysis to Fc proteins. C1q (left panel) and C5-9
deposition (right panel) to Fc-fusions determined by ELISA. Each point represents the
mean optical density (+/-SD) of duplicate wells for each mouse within a given group.
Data from one of three replicate experiments are shown.
Fig 5: Hexameric Fc protein protects against platelet loss in a mouse model of ITP.
Balb/C mice were injected i.p. with Hexa-Fc, IVIG (GammaGard), or PBS. One hour
later ITP was induced in all mice. Platelets were enumerated at indicated time-points
post treatment.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
According to a first aspect of the invention, a method for treatment of a mammalian
subject for an autoimmune or inflammatory disease is provided. The subject is treated
by administering a polymeric protein comprising five, six or seven polypeptide monomer
units; wherein each polypeptide monomer unit comprises an Fc receptor binding portion
comprising two immunoglobulin G heavy chain constant regions.
The term "immunoglobulin G heavy chain constant region" means a native
immunoglobulin G heavy chain region, or variant or fragment thereof. The Fc receptor
binding portion typically comprises the Fc portion of an immunoglobulin G, or fragment or
variant thereof. The term "Fc portion" includes a fragment of an IgG molecule which is
obtained by limited proteolysis with the enzyme papain, which acts on the hinge region of
IgG. An Fc portion obtained in this way contains two identical disulphide linked peptides
containing the heavy chain CH2 and CH3 domains of IgG, also referred to as Cy2 and
Cy3 domains respectively. The two peptides are linked by two disulphide bonds
between cysteine residues in the N-terminal parts of the peptides. The arrangement of
the disulphide linkages described for IgG pertain to natural human antibodies. There
may be some variation among antibodies from other mammalian species, although such
antibodies may be suitable in the context of the present invention. Antibodies are also
found in birds, reptiles and amphibians, and they may likewise be suitable. Nucleotide
and amino acid sequences of human Fc IgG are disclosed, for example, in Ellison et al.
(1982) NUCLEIC ACIDS RES. 10: 4071-4079. Nucleotide and amino acid sequences of
murine Fc lgG2a are disclosed, for example, in Bourgois et al. (1974) EUR. J.
BIOCHEM. 43: 423-435. Immunoglobulin G heavy chain constant regions may typically
be produced by recombinant expression techniques, and associate as monomer units by
disulphide linkages, as occurs in native antibodies. Alternatively, the two constant
regions may be produced as a single amino acid chain with an intervening linker region,
i.e. as a single chain Fc (scFc) typically also by recombinant expression techniques.
scFc molecules are described in US 201 1/0081345, including examples having the
following general structure from N to C terminus: Hinge-CH2-CH3-linker-Hinge-CH2-
CH3.
Typically, each of the immunoglobulin G heavy chain constant regions comprises an
amino acid sequence of a mammalian heavy chain constant region, preferably a human
heavy chain constant region; or variant thereof. A suitable human IgG subtype is IgG
The Fc receptor binding portion may comprise more than the Fc portion of an
immunoglobulin. For example, it may include the hinge region of the immunoglobulin
which occurs between CH1 and CH2 domains in a native immunoglobulin. For certain
immunoglobulins, the hinge region is necessary for binding to Fc receptors. Preferably,
the Fc receptor binding portion lacks a CH1 domain and heavy chain variable region
domain (VH). The Fc receptor binding portion may be truncated at the C- and/or Nterminus
compared to the Fc portion of the corresponding immunoglobulin. Such a Fc
receptor binding portion is thus a "fragment" of the Fc portion.
The polymeric protein is formed by virtue of each immunoglobulin G heavy chain
constant region comprising 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. Because IgM and IgA are naturally polymeric, whereas IgG
is naturally monomeric, the ability of monomer units based on IgG heavy chain constant
regions to form polymers may be improved by modifying the parts of the IgG heavy chain
constant regions to be more like the corresponding parts of IgM or IgA. Suitably, each of
the immunoglobulin heavy chain constant regions or variants thereof is an IgG heavy
chain constant region comprising an amino acid sequence which comprises a cysteine
residue at position 309 according to the EU numbering system, and preferably also a
leucine residue at position 310. The EU numbering system for IgG is described in Kabat
EA et al, 1983 Sequences of proteins of immunological interest. US Department of
Health and Human Services, National Institutes of Health, Washington DC. Sorensen et
al (1996) J Immunol 156: 2858-2865 describes the mutation of Leu 309 to Cys 309 in a
human lgG3 molecule comprising a IgM tailpiece, to promote polymer formation. Leu
309 corresponds by sequence homology to Cys 414 in C 3 domain of IgM and Cys 309
in the Ca2 domain of IgA. (Note that the numbering of amino acid residues differs
between the IgM and IgA or IgG classes.) Other mutations may also be advantageous.
Suitably, each polypeptide monomer unit comprises a tailpiece region fused to each of
the two immunoglobulin G heavy chain constant regions; wherein the tailpiece region of
each polypeptide monomer unit facilitates the assembly of the monomer units into a
polymer. Typically, the tailpiece region is fused C-terminal to each of the two
immunoglobulin heavy chain constant regions. Suitably, the tailpiece region is an IgM or
IgA tailpiece, or fragment or variant thereof. These tailpieces are the final portions of the
C 4 domain of IgM or the Ca3 domain of IgA respectively.
Where a region is described as being fused C-terminal to another region, the former
region may be fused directly to the C-terminus of the latter region, or it may be fused to
an intervening amino acid sequence which is itself fused to the C-terminus of the latter
region. N-terminal fusion may be understood analogously.
An intervening amino acid sequence may be provided between the heavy chain constant
region and the tailpiece, or the tailpiece may be fused directly to the C-terminus of the
heavy chain constant region. For example, a short linker sequence may be provided
between the tailpiece region and immunoglobulin heavy chain constant region. Typical
linker sequences are of between 1 and 20 amino acids in length, typically 2, 3, 4, 5, 6 or
up to 8, 10, 12, or 16 amino acids in length. A suitable linker to include between the
heavy chain region and tailpiece region encodes for Leu-Val-Leu-Gly.
A preferred tailpiece region is the tailpiece region of human IgM, which is
PTLY VSLVMSDTAGTCY (Rabbitts TH et al, 1981. Nucleic Acids Res. 9 (18), 4509-
4524; Smith et al (1995) J Immunol 154: 2226-2236). Suitably, this tailpiece may be
modified at the N-terminus by substituting Pro for the initial Thr, thus generating the
sequence PPLYNVSLVMSDTAGTCY. This does not affect the ability of the tailpiece to
promote polymerisation of the monomer. Further suitable variants of the human IgM
tailpiece are described in Sorensen et al (1996) J Immunol 156: 2858-2865. A further
IgM tailpiece sequence is GKFTLYNVSLIMSDTGGTCY from rodents (Abbas and
Lichtman, Cellular and Molecular Immunology, Elsevier Saunders, 5th Edn, 2005). An
alternative preferred tailpiece region is the tailpiece region of human IgA, which is
PTHVNVSWMAQVDGTCY (Putnam FW et al, 1979, J. Biol. Chem 254: 2865-2874)]
Other suitable tailpieces from IgM or IgA of other species, or even synthetic sequences
which facilitate assembly of the monomer units into a polymer, may be used. It is not
necessary to use an immunoglobulin tailpiece from the same species from which the
immunoglobulin heavy chain constant regions are derived, although it is preferred to do
so.
"Variants" and "fragments" are as defined in relation to heavy chain constant regions. A
variant of an IgM tailpiece typically has an amino acid sequence which is identical to
PPLYNVSLVMS DTAGTCY in 8, 9, 10, 11, 12, 13, 14 ,15, 16 or 17 of the 18 amino acid
positions. A variant of an IgA tailpiece typically has an amino acid sequence which is
identical to PTHVNVSWMAQVDGTCY in 8, 9 , 10, 11, 12, 13, 14 ,15, 16 or 17 of the 18
amino acid positions. Fragments of these IgM or IgA tailpieces typically comprise 8, 9,
10, 11, 12, 13, 14 ,15, 16 or 17 amino acids. Fragments of variants are also envisaged.
Typically, fragments and variants of the IgM or IgA tailpiece retain the penultimate
cysteine residue, as this is believed to form a disulphide bond between two monomer
units in a polymeric protein.
The ability of a given tailpiece region to facilitate assembly of the monomer units into a
polymer may be tested by comparing the proportion of protein having a high molecular
size when formed from monomer units lacking a tailpiece with monomer units comprising
a tailpiece. The latter may form a higher proportion of high molecule size polymers
under native conditions. Native molecular weights can be determined by size-exclusion
chromatography, for example on Sephadex-200 columns on an AKTA FPLC
(Amersham). Alternatively, non-reducing gel electrophoresis may be used, as described
in Smith et al (supra) or Sorensen et al (supra).
When the monomer units have assembled into a polymer, the Fc receptor binding
portions are arranged in a planar polymeric structure which is spatially orientated to allow
each Fc receptor binding portion to bind to an Fc receptor. IgM is naturally pentameric or
hexameric and IgA naturally forms dimers, trimers or tetramers. These properties
appear to be determined, at least in part, by the ability of the tailpiece to cause the
monomers to associate into polymers. Pentameric IgM is formed when the IgM
associates with the J chain, although it is typically hexameric in the absence of the J
chain. The J chain may or may not be included as a further component of the polymeric
fusion protein of the invention. Production is simplified by omitting the J chain
polypeptide, which in any case, is not needed for polymerisation of IgG (Ghumra et al,
2008). Secretory IgM or IgA found at mucosal surfaces contains secretory component
(SC), part of the polymeric Ig-receptor used to translocate them from blood to secretions.
The SC may or may not be included as a further component of the polymeric protein of
the invention. Production is simplified by omitting the SC which, in any case, is not
needed for polymerisation.
The polymeric protein comprising five, six or seven polypeptide monomer units is used
according to the first aspect of the invention. The exemplary Fc protein comprising
human lgG1 heavy chain constant regions and a human IgM tailpiece described herein,
forms hexamers and dimers. Variants based on other IgG heavy chain constant regions,
including different tailpieces or no tailpiece, may form polymers having different numbers
of monomers. In particular, they may be pentamers or heptamers rather than hexamers.
Where Fc proteins naturally associate into polymers having different numbers of
monomer units, polymers having the required number of monomer units can be
separated according to molecular size, for example by gel filtration. Mixtures where at
least a proportion of the protein is in the form of a pentamer, hexamer and/or heptamer
may also be used. Suitably pentamers, hexamers and/or heptamers are used in the
absence of proteins having other numbers of monomer units.
In the polymeric proteins to be used according to the first aspect of the invention, the Fc
receptor binding portion of each monomer unit is capable of binding to an Fc receptor. It
will be appreciated that Fc receptor binding portions which comprise the Fc portion of a
particular immunoglobulin, will bind to different Fc receptors depending on the binding
specificity of the particular immunoglobulin. Typically, the Fc receptor binding portion will
have an affinity for a given Fc receptor which is at least comparable to the affinity of a
native monomeric immunoglobulin molecule. However, lower affinities may be tolerated
because the polymeric protein comprises multiple such Fc receptor binding portions, and
will therefore bind to Fc receptors with higher avidity. Therefore, the Fc receptor binding
portion will typically have an affinity which is at least a tenth, suitably at least a fifth and
most suitably at least a half of the affinity of the corresponding native monomeric
immunoglobulin molecule which binds to the given Fc receptor.
Affinity constants can be readily determined by surface Plasmon Resonance Analysis
(Biacore). The Fc receptor binding portions can be passed over flow cells from CM5
sensor chips amine-coupled to Fc receptors. Equimolar concentrations of the Fc
receptor binding portion or intact monomeric antibody may be injected over each Fc
receptor and association and dissociation observed in real time. Data from a BIAcore X
or 3000 machines may be analyzed using BIAevaluation 3.0 software to determine
accurate affinity constants.
There are three classes of human Fey receptor (Gessner et al (1998) Ann Hematol 76:
231-48; Raghavan and Bjorkman (1996) Ann Rev Cell Dev Biol 12: 181-220). FcyRI
(CD64) binds monomeric IgG with high affinity. FcyRI I (CD32) and FcyRI 11(CD16) are
the low affinity receptors for Fc and can only interact with high affinity with antibodies that
are presented to the immune system as immune complexes (ICs). Although larger (>350
kDa) multimeric IgG and circulating ICs are mostly removed in the preparation of IVIG,
these are common in healthy individuals where they can contribute as much as 10% to
the overall plasma Ab concentration, indicating a physiological role in maintaining
immune homeostasis in these healthy individuals (Nezlin R (2009) Immunol Lett
122,141-4).
FcyRII and FcyRIII are closely related in the structure of their ligand-binding domains. In
humans three separate genes, FcyRIIA, FcyRIIB, and FcyRIIC, two of which give rise to
alternatively spliced variants, code for FcyRII. FcyRlla delivers activating signals
whereas FcyRllb delivers inhibitory signals. The functional basis for the divergent
signals arises from signaling motifs located within the cytoplasmic tails of the receptors.
An immunoreceptor tyrosine-based inhibitor motif (ITIM) located in the cytoplasmic tail of
the FcyRllb is involved in negative receptor signaling. The ITIM motif is a unique feature
of the FcyRllb receptor as it is not apparently present in any other Fey receptor class. In
contrast, an activatory immunoreceptor tyrosine-based activation motif or ITAM is located
in the cytoplasmic tail of FcYRIIa. ITAM motifs transduce activating signals. They are
also found in the FcRy-chains, which are identical to the g chains of the high affinity IgE
receptor (FCERI). While FcYRIIa and FCYRI ID are widely expressed on myeloid cells and
some T-cell subsets they are notably absent from N cells. There are two alleles for the
FcYRIIa receptor in humans, referred to as His131 (H131) and Arg131 (R131). The
FcYRIIa-Arg131 allele is associated with increased susceptibility to infection by
encapsulated bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae and
Neiserria meningitidis, which elicit lgG2 responses (Pleass RJ & Woof JM, 2001). The Fc
receptor encoded by this allele cannot bind lgG2 and is therefore unable to elicit
clearance of lgG2-coated bacteria. Both variants bind human lgG1, however.
Human FCYRI II is also present in multiple isoforms derived from two distinct genes
(FCYRII IA and FCYRII IB) . FcYRIIIb is unique in its attachment to the cell membrane via a
glycosylphosphatidyl anchor. FcYRIIIb expression is restricted to neutrophils while
FcyRIIIa is expressed by macrophages, and NK cells. FcYRIIIa is also expressed by
some T-cell subsets and certain monocytes. FCYRI Ila requires the presence of the
FcRY-chain or the Ώ3z- \h for cell surface expression and signal transduction. The
FcRY-chain and the 3z- h ίh are dimeric and possess ITAM motifs. FcYRIIIa forms a
multimeric complex with these subunits and signalling is transduced through them.
Thus, there is considerable FCYR receptor heterogeneity and diverse expression
The binding sites for FCYRI I and FCYRIII map to the hinge and proximal region of the CH2
domain of IgG, the same region originally identified for FCYRI (Duncan et al (1988)
Nature 332: 563-4; Morgan et al (1995) Immunol 86: 319-324; Lund et al (1991) J
Immunol 147: 2657-2662).
FCY receptors (FCYRS) trigger activatory and/or inhibitory signalling pathways that set
thresholds for cell activation and culminate in a well-balanced immune response
(Nimmerjahn F & Ravetch JV (2008) Nat. Rev. Immunol. 8: 34-47). Activating and
inhibitory FcRs are widely expressed throughout the haematopoetic system but
particularly on professional antigen presenting cells (APCs) (Nimmerjahn F & Ravetch JV
(2008) supra). For example in humans, FCYRI is constitutively expressed by blood
myeloid dendritic cells (DCs) and FCYRII has been detected on every DC subset
examined to date, whereas the expression of FCYRI , FCYRIIB and FCYRI II dominate on
murine DCs (Ravetch JV (2003) in Fundamental Immunology (ed. Paul WE) 685-700
(Lippincott-Raven, Philidelphia); Bajtay Z et al (2006) Immunol. Lett. 104: 46-52). FCYRS
also play a pre-eminent role in antigen presentation and immune-complex-mediated
maturation of dendritic cells (DCs), and in regulation of B-cell activation and plasma-cell
survival (Ravetch JV (2003) supra; Bajtay Z et al, (2006) supra). Moreover, by regulating
DC activity, FcyRs control whether an immunogenic or tolerogenic response is initiated
after the recognition of antigenic peptides presented on the surface of DCs to cytotoxic T
cells, T helper cells, and regulatory T cells. FcyRs also co-operate with Toll-like
receptors (TLRs) in controlling levels of the important regulatory cytokines, IL-12 and IL-
10 (Polumuri SK, 2007, J. Immunol 179: 236-246). Thus, FcyRs are involved in
regulating innate and adaptive immune responses, which makes them attractive targets
for the development of novel immunotherapeutic approaches (Nimmerjahn F & Ravetch
JV (2008) supra).
It is known that the inhibitory FcyRIIB controls the magnitude of the immune response,
as DCs derived from FcyRI IB-knockout mice generate stronger and longer-lasting
immune responses in vitro and in vivo (Bergtold A, Desai DD, et al (2005) Immunity 23:
503-514; Kalergis A M & Ravetch JV. (2002) J. Exp. Med. 195: 1653-1659). More
importantly, FcyRI IB-deficient DCs or DCs incubated with a mAb that blocks immune
complex binding to FcyRIIB showed a spontaneous maturation (Boruchov AM, et al
(2005) J. Clin. Invest. 115: 2914-2923; Dhodapkar KM, t al (2005) Proc. Natl Acad. Sci.
USA 102: 2910-2915). This suggests that the inhibitory FcyR not only regulates the
magnitude of cell activation but also actively prevents spontaneous DC maturation under
non-inflammatory steady-state conditions. Indeed, low levels of immune complexes can
be seen in the serum of healthy donors, emphasizing the importance of regulatory
mechanisms that prevent unwanted DC activation (Dhodapkar KM, et al (2005) Proc.
Natl Acad. Sci. USA 102: 2910-2915). The loss of FcyRIIB also results in the priming of
more antigen-specific T cells (Kalergis A M & Ravetch JV. (2002) J. Exp. Med. 195:
1653-1659). Therefore the binding of the polymeric Fc protein to multiple copies of
FcyRIIB may induce negative responses from cells expressing this receptor.
FcRL5 is a recently described Fc receptor capable of inhibitory signalling, which is
expressed on B cells and which binds aggregated IgG but not monomeric IgG (Wilson et
al, 2012). These authors propose that IgG may bind cooperatively to FcRL5 and FcyRllb
co-expressed on B cells. As described in Example 3, an exemplary hexameric protein
can bind FcRL5 independently of FcyRllb.
Where the Fc receptor binding portion comprises immunoglobulin heavy chain constant
regions of a human IgG isotype or variants thereof, it will typically bind to human Fcyreceptors
(FcyRI, FcyRII and FcyRIII) and/or human FcRL5. Surface Plasmon
Resonance Analysis as described above can be used to determine affinity constants.
Typical affinity constants for binding of human lgG1 or lgG3 to FcyRI are about 10 M;
for FcyRII are about 0.6-2.5x1 0 6 M; for FcyRIIIA are about 5x1 0"5 M; for FcyRIIIB are
about 0.6-2.5x1 0 6 M. Monomeric IgG does not noticeably bind to FcRL5, although
FcRL5 does bind with high avidity to aggregated IgG, suggesting FcRL5 is a low to
medium affinity receptor for monomeric IgG with an affinity constant of about 10 -10 6 M
(taken from Wilson et al, 2012). Alternatively, ELISAs may be used essentially as
described in Example 3 to obtain a semi-quantitative indication of binding properties.
Fc receptor binding portions also typically bind to lectins, also referred to as glycan
receptors, particularly lectins which bind to sialic acid. Exemplary lectins are human DCSIGN
(or mouse homologue SIGN-R1) and CD22, which contribute to the therapeutic
properties of IVIG (reviewed in Mekhaiel, 201 1b). Polymeric proteins are predicted to
interact with these receptors, as explained in Example 3. SIGN-R1 binding can be
determined by surface plasmon resonance as described in Jain et al, 2012. Analogous
methods may be used to determine binding to CD22 or DC-SIGN. Human 2,6 sialylated
Fc binds SIGN-R1 and human DC-SIGN with affinity constants 2.7x1 0 6 M and 3.6x1 0 6
M respectively (Anthony et al, 2008, PNAS 105: 19571-19578). sialylated Fc does not
bind appreciably. Multimeric sialoproteins e.g. haemagglutinin are known to bind to sialic
acid receptors with affinities in the order of 10 M compared to monomers which bind 10
2M (Mammen M, Choi SK, Whitesides GM (1998) Angew Chem Int Edit 37: 2755-2794.)
Hence, as an increase in avidity can enhance an inherently weak substrate affinity,
presenting terminal sialic acid residues on polymeric proteins would likewise be expected
to significantly enhance binding to sialic acid receptors.
The appropriate limits for and determination of affinity constants for Fc receptor binding
portions which comprise variants of native immunoglobulin heavy chain constant regions,
or fragments of Fc portions, is as described above.
A "variant" refers to a protein wherein at one or more positions there have been amino acid
insertions, deletions, or substitutions, either conservative or non-conservative.
A "variant" may have modified amino acids. Suitable modifications include acetylation,
glycosylation, hydroxylation, methylation, nucleotidylation, phosphorylation, ADPribosylation,
and other modifications known in the art. Such modifications may occur
postranslationally where the peptide is made by recombinant techniques. Otherwise,
modifications may be made to synthetic peptides using techniques known in the art.
Modifications may be included prior to incorporation of an amino acid into a peptide.
Carboxylic acid groups may be esterified or may be converted to an amide, an amino group
may be alkylated, for example methylated. A variant may also be modified posttranslationally,
for example to remove carbohydrate side-chains or individual sugar moieties
e.g. sialic acid groups or to add sialic acid groups.
By "conservative substitutions" is intended combinations such as Val, lie, Leu, Ala, Met;
Asp, Glu; Asn, Gin; Ser, Thr, Gly, Ala; Lys, Arg, His; and Phe, Tyr, Trp. Preferred
conservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys,
Arg; and Phe, Tyr.
Typical variants of the immunoglobulin G heavy chain constant regions will have an
amino acid sequence which is at least 70%, typically at least 80%, at least 90%, at least
95%, at least 99% or at least 99.5% identical to the corresponding immunoglobulin G
heavy chain constant region of a native immunoglobulin. Suitably, the variant is a variant
of the human immunoglobulin G 1 heavy chain constant region and has an amino acid
sequence which is at least 90%, at least 95%, at least 99% or at least 99.5% identical to
the latter.
A "fragment" refers to a protein wherein at one or more positions there have been deletions.
Typically a fragment of a Fc portion comprises at least 60%, more typically at least 70%,
80%, 90%, 95% or up to 99% of the complete sequence of the Fc portion. Fragments of
variants are also encompassed.
The percent sequence identity between two polypeptides may be determined using
suitable computer programs, for example the GAP program of the University of
Wisconsin Genetic Computing Group and it will be appreciated that percent identity is
calculated in relation to polypeptides whose sequence has been aligned optimally.
The alignment may alternatively be carried out using the Clustal W program (Thompson
et a/., (1994) Nucleic Acids Res., 22(22), 4673-80). The parameters used may be as
follows:
• Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap
penalty; 3, number of top diagonals; 5. Scoring method: x percent.
· Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.
• Scoring matrix: BLOSUM.
Variants may be natural or made using the methods of protein engineering and site-directed
mutagenesis as are well known in the art.
"Peptides" generally contain up to 10, 20, 50 or 100 amino acids. Peptides and
polypeptides may conveniently be blocked at the N- or C-terminus so as to help reduce
susceptibility to exoproteolytic digestion. Peptides and polypeptides may be produced by
recombinant protein expression or in vitro translation systems (Sambrook et al,
"Molecular cloning: A laboratory manual", 2001 , 3rd edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY). Peptides may be synthesised by the Fmocpolyamide
mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org.
Chem. 46, 3433 and references therein.
Suitably, in the Fc protein for use according to the first aspect of the invention, each of
the immunoglobulin G heavy chain constant regions comprises an amino acid sequence
which is modified compared to the amino acid sequence of a native immunoglobulin G
heavy chain constant region, to modify the affinity of the Fc receptor binding portion for at
least one Fc receptor. Typically the affinity of the Fc receptor binding portion for a low
affinity inhibitory and/or activatory Fey receptor may be increased.
The interactions between IgG and Fc receptors have been analyzed in biochemical and
structural studies using wild type and mutated Fc. One consensus indicates that some
regions for binding to Fc receptors are located in the part of the hinge region closest to
the CH2 domain and in the amino-terminus of the CH2 domain that is adjacent to the
hinge, including for example residues 233-239 (Glu-Leu-Leu-Gly-Gly-Pro-Ser). Mutations
within this region can result in altered binding to Fc receptors. This region appears to be
responsible for some of the direct interactions with Fc receptors (Woof JM & Burton D,
Nature Reviews Immunology 2004, 4: 89-99). Further into the CH2 domain, and away
from the hinge, are other residues that may, at least in some contexts, contribute to Fc
receptor binding, including for example, Pro-329 of human lgG1 (EU numbering) which
appears to be involved in direct contact with the Fc receptor and Asn-297 which appears
to be the sole site for N-linked glycosylation within the Fc region of human lgG1. The
presence of carbohydrate at this residue may contribute to the binding to Fc receptors.
Activatory Fey receptors are as described above; in the human, the low affinity activatory
Fey receptors are FcyRIIA/C and FcyRIIIA. FcyRI is the high affinity activatory receptor.
FcyRIIB is an inhibitory human Fc receptor. As the ligand binding properties of FcyRIIB
and FcyRIIA/C are the same, it may not be possible to increase affinity for FcyRIIA/C
whilst simultaneously decreasing affinity for FcyRIIB. Lazar et al (2006) PNAS 103:
4005-10 describes mutations in the Fc portion of a human IgG which affect binding
affinity to different Fc receptors. A wildtype IgG bound to FcyRllla with a KD of 252 nM;
the KD of a I332E mutant was 30 nM and the K of a S239D/I332E mutant was 2 nM.
Combination of an A330L mutation with S239D/I332E increased FcyRllla affinity and
reduced FcyRllb affinity. Shields RL et al (2001) J. Biol. Chem. 276: 6591-6604
describes mutations in the Fc portion of human lgG1 which affect binding affinity to
different Fc receptors. The S298A mutation increased affinity for FcyRllla and
decreased affinity for FcyRIIA; the E333A mutation increased affinity for FcyRllla and
decreased affinity for FcyRIIA; the mutation K334A increased affinity for FcyRllla. Any
or all of the above mutations may be used individually or in combination. Other suitable
mutations may be identified by routine methods.
The affinity of the Fc receptor binding portion for CD22 or DC-SIGN/SIGN-R1 may be
increased by increasing the amount of bound sialic acid on the Fc receptor binding
portion. Typically this is achieved by increasing the amount or proportion of terminal
sialic acid residues on the N297 glycan, which may be added post translationally to the
Fc peptide in the endoplasmic reticulum.
Other mutations may suitably be made to improve the efficacy of the Fc receptor binding
portions. Suitably, each of the immunoglobulin G heavy chain constant regions
comprises an amino acid sequence which is modified compared to the amino acid
sequence of a native immunoglobulin G heavy chain constant region, to increase the in
vivo half life of the polymeric protein, suitably by increasing the affinity of the Fc receptor
binding portion for neonatal Fc receptor. Increasing the serum persistence allows higher
circulating levels, less frequent administration and reduced doses. This can be achieved
by enhancing the binding of the Fc region to neonatal FcR (FcRn). FcRn, which is
expressed on the surface of endothelial cells, binds the Fc in a pH-dependent manner
and protects it from degradation. Although hexameric-Fc described in the Examples was
unable to bind human FcRn it did bind very well to mouse FcRn. The amino acid
substitutions M252Y/S254T/T256E and/or H433K/N434F may be introduced into the Fcreceptor
binding portion to increase in vivo half life of IgG without unduly affecting FcyR
interactions (Vaccaro C, er al (2005) Nat. Biotech. 23: 1283-1288). In addition or in the
alternative, H310 may be reintroduced.
The binding of multiple Fc receptors by a polymeric Fc protein may cause different
intracellular signalling phenomena than the binding of a single Fc receptor by a
monomeric Fc protein or IgG. Binding of multiple Fc receptors by the polymeric protein
may be more effective in blocking the binding of the Fc receptors to other ligands,
particularly in the case of low affinity Fc receptors. The efficacy of the polymeric protein
can be compared against the efficacy of a monomeric unit which does not form
polymers, in many ways. In such tests, it is typical for the monomeric units of the
polymeric Fc protein to, individually, have the same affinity for a given Fc receptor as the
monomeric unit which does not form polymers.
Polymeric proteins will have greater avidity for low affinity receptors including Fc
receptors than will the control monomeric units. They may also have a greater avidity for
FcRL, FcRn, CD22, DC-SIGN or other Fc-receptors. Avidity is the overall binding
strength of a polyvalent interaction. The interaction between a Fc binding region and a
Fc receptor has a characteristic affinity, whereas the avidity of the interaction increases
almost geometrically for each interaction. For low affinity Fc receptors, the increase in
binding strength may allow a biologically relevant interaction with a polymeric protein,
which could not be achieved by a monomeric or dimeric unit. Multivalent binding by
polymeric proteins results in a considerable increase in stability as measured by the
equilibrium constant ( LJmol), compared to binding of a control monomeric protein. For
example, a typical monovalent interaction between an Fc portion and an Fc receptor may
have an equilibrium constant of about 04 L/mol. A hexavalent interaction may provide
for an equilibrium constant of about 10 1 L/mol. The equilibrium constant may vary
depending on the Fc portion and the Fc receptor. However, a hexameric protein will
typically exhibit an increase in the binding energy compared to a control monomeric
protein of up to about 104, 105 or 106 fold, or even greater than 106 fold. Similar
increases in binding energy and equilibrium constants may be expected for multivalent
interactions with the FcRL5, FcRn, CD22 or DC-SIGN. For description of avidity and
affinity see textbook Immunology by Roitt, Brostoff and Male, 2nd edition 1989.
The avidity for Fc receptors of the polymeric protein may be compared to that of the
monomeric unit which does not form polymers by Surface Plasmon Resonance Analysis
(Biacore), as described above.
It has been found that fusion of a bulky antigen to the polymeric protein can diminish the
ability of the polymeric protein to bind to Fc receptors (Mekhaiel et al, 201 1a). Suitably,
the polymeric protein for use according to the first aspect of the invention does not
comprise (for example by being fused or conjugated to) a moiety which reduces binding
avidity for FcyRII. The effect on avidity can be determined by comparing binding avidity
for a given receptor by the polymeric protein comprising the moiety to be tested with the
avidity achieved by the polymeric protein lacking the moiety. Typically, if the avidity is
reduced by more than 10-fold, or more than 5-fold or more than 2-fold, the polymeric
protein comprising the moiety may not be suitable. Reduced binding avidity for FcyRI I
would also be indicative of reduced binding avidity for other Fc receptors, including FcyRI
or FCYRI II . In the alternative, binding avidity for either or both of these other receptors
could be tested.
Increased avidity of polymeric proteins for low-affinity receptors, as described in Example
3, may engage the biological mechanisms underlying IVIG therapy, particularly those
mediated by low-affinity receptors FcyRIIB, FcyRIIIA, CD22 and DC-SIGN. Increased
avidity for the high affinity IgG receptor FcyRI was also observed for the exemplary
hexameric protein. FcyRI is implicated in inflammatory autoimmune disease (Hussein
OA et al, Immunol Invest 2010, 39:699-712) and FcyRI-directed immunotoxins inhibit
arthritis (Van Vuuren AJ et al, J. Immunol 2006 176: 5833-8). Also MicroRNA-127
inhibits lung inflammation by targeting FcyRI (Xie T et al, J. Immunol 2012 188, 2437-
44). Moreover, ligation of FcyRI on macrophages has been proposed to induce
production of the anti-inflammatory cytokine IL-10 in US 2004/0062763 (Temple
Univeristy; Mosser). Thus, ligation of FcyRI may also have a beneficial effect in the
context of the present invention, although FcyRI is not thought to underlie the effects of
IVIG.
The immunomodulatory properties of the polymeric protein arise from interactions
between the Fc receptor binding portions and Fc receptors and/or other receptors and
components of the immune system which interact with Fc portions. The polymeric
protein for use according to the first aspect of the invention does not comprise a further
immunomodulatory portion; or an antigen portion that causes antigen-specific
immunosuppression when administered to the mammalian subject. Contrary to
therapeutic approaches which provide immunosuppressive agents such as TNFreceptor,
the polymeric proteins described herein rely on a different therapeutic
mechanism, which may make them more widely useful in therapy. Neither do the
polymeric proteins rely on an antigen-specific immunosuppressive effect, which limits the
application of other therapeutic approaches to specific diseases.
An "immunomodulatory portion" is an agent which has an immunomodulatory activity in a
healthy or diseased mammalian subject when covalently linked to a polymeric protein as
described herein, or when present in the absence of said polymeric protein.
"Immunomodulatory activity" refers to altering an immune response in a subject, to
increase or decrease components of the immune system such as cytokines or
antibodies; or to increase or decrease immune functions such as antigen presentation.
An "immunomodulatory" portion may be a chemokine or chemokine receptor, cytokine or
cytokine receptor, Toll like receptor (TLR), acute phase protein, complement component,
immune receptor, CD molecule, or signal transduction molecule, for example. Such
agents are known to possess immunomodulatory activities in a healthy or diseased
mammalian subject in the absence of the polymeric protein. Examples of such agents
are described in Immunology textbooks such as (Abbas and Lichtman, Cellular and
Molecular Immunology, Elsevier Saunders, 5th Edn, 2005), and the skilled worker can
find further examples in relevant literature. Thus, conjugates or fusion proteins
comprising any of these agents and the polymeric protein are excluded from the
invention. Immunomodulatory portions found in monomeric Fc fusion proteins known as
etanercept, alefacept, abatacept, belatacept, atacicept, briobacept, rilonacept or
afilbercept are particularly excluded. Antibody fragments such as Fab or scFvs may also
be regarded as "immunomodulatory portions". Such fragments may bind to components
of the immune system, thus having an immunomodulatory activity. When fused or
conjugated to monomeric units of the polymer protein, antibody fragments may
recapitulate the complete antibody structure i.e. Fc and antigen-combining regions.
Such agents may have a more pronounced immunomodulatory activity when covalently
linked to the polymeric protein, than when present in isolation. Immunomodulatory
portions found in therapeutic antibodies, such as the antibody known as Remicade which
binds TNFa, are particularly excluded. Suitably, the polymeric protein does not comprise
any antibody portions other than the immunoglobulin G heavy chain constant regions
and optionally hinge region, whether immunomodulatory or not.
As noted above, the immunomodulatory properties of the polymeric protein arise from
interactions between the Fc receptor binding portions and Fc receptors and/or other
receptors and components of the immune system which interact with Fc portions.
Modifications of the Fc receptor binding portions, such as modified glycosylation, which
modify binding to Fc receptors, lectins or other immune system components are not
regarded as further immunomodulatory portions, but as components of the Fc receptor
binding portions. Therefore, they are not excluded from the invention.
A suitable experimental test for an immunomodulatory portion is to provide a polymeric
protein lacking the putative immunomodulatory portion and a polymeric protein
comprising the putative immunomodulatory portion and test either protein for efficacy in
the mouse model of ITP described in Example 4. If the putative immunomodulatory
portion possesses an immunomodulatory activity, it may alter parameters of the
response. For example, it may alter the rate or extent of platelet recovery.
An "antigen" is a molecule that binds specifically to an antibody or a TCR. Antigens that
bind to antibodies include all classes of molecules, and are called B cell antigens.
Exemplary types of molecule include peptides, polypeptides, glycoproteins,
polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments thereof,
portions thereof and combinations thereof. TCRs bind only peptide fragments of proteins
complexed with MHC molecules; both the peptide ligand and the native protein from
which it is derived are called T cell antigens. "Epitope" refers to an antigenic determinant
of a B cell or T cell antigen. Where a B cell epitope is a peptide or polypeptide, it
typically comprises three or more amino acids, generally at least 5 and more usually at
least 8 to 10 amino acids. The amino acids may be adjacent amino acid residues in the
primary structure of the polypeptide, or may become spatially juxtaposed in the folded
protein. T cell epitopes may bind to MHC Class I or MHC Class I I molecules. Typically
MHC Class l-binding T cell epitopes are 8 to 11 amino acids long. Class II molecules
bind peptides that may be 10 to 30 residues long or longer, the optimal length being 12
to 16 residues. Peptides that bind to a particular allelic form of an MHC molecule contain
amino acid residues that allow complementary interactions between the peptide and the
allelic MHC molecule. The ability of a putative T cell epitope to bind to an MHC molecule
can be predicted and confirmed experimentally (Dimitrov I et al, Bioinformatics. 2010 Aug
15;26(16):2066-8).
The polymeric protein for use according to the first aspect of the invention does not
comprise an antigen portion that causes antigen-specific immunosuppression when
administered to the mammalian subject. By "comprise", we include antigens which are
covalently linked to at least one of the peptide monomer units, such as by chemical
conjugation or recombinant fusion. Typically, the polymeric protein does not comprise a
portion that acts as an antigen, whether by causing immunosuppression or
immunostimulation, when administered to the mammalian subject.
By "antigen-specific immunosuppression", we include suppression of antibody responses
and/or suppression of T cell responses. T cell responses may be suppressed by clonal
deletion of antigen-reactive T cells, anergy or suppression, such as via induction of
regulatory T cells. T cell responses may also be deviated from more aggressive to less
aggressive forms, for example from Th1 type responses to Th2 type responses.
A suitable test for an antigen portion that causes antigen-specific immunosuppression is
to provide a polymeric protein lacking the antigen portion and a polymeric protein
comprising the antigen portion and testing either protein in the mammalian subject to be
treated for the autoimmune or inflammatory disease. Polymeric proteins lacking or
containing the antigen may be administered sequentially in the same mammalian
subject, and the subject monitored for an immune response to the antigen following
treatment with either protein. Alternatively, groups of like subjects may be treated either
with the polymeric protein lacking the antigen or the polymeric protein comprising the
antigen. Conventional techniques may be used to identify and monitor the immune
response to the antigen, whether a B cell response or a T cell response. If there is no
difference in the extent or type of immune response to the antigen in the subjects
following administration of the polymeric protein lacking the antigen, compared to the
polymeric protein comprising the antigen, then the antigen does not cause antigenspecific
immunosuppression when administered to the mammalian subject. Thus,
antigen-specific means of immunosuppression resulting from an antigen portion of a
polymeric protein are excluded. That does not mean that the immune response to a
particular antigen may not decrease in response to therapy. The immune response to an
auto-antigen implicated in an autoimmune disease may decrease in response to therapy,
but this can be achieved by the polymeric protein lacking the antigen equally as well as
by a polymeric protein comprising the antigen. Analogous tests may be used to identify
whether the polymeric protein comprises a portion that acts as an antigen when
administered to the mammalian subject. In such tests, dose, formulation and features of
administration of the polymeric proteins are the same to allow a meaningful comparison.
Alternatively, polymeric proteins may be tested in healthy mammalian subjects to
determine whether a portion that acts as an antigen is present. Such tests are
particularly useful to identify antigen-specific immunostimulation. Suitably, antigenspecific
immunostimulation does not occur. Such tests may be useful to confirm nonimmunogenicity
of the polymeric protein. Components of the polymeric protein that are
not naturally found in the mammalian subject to which the protein is to be administered,
such as linker peptides, can be tested for immunogenicity and non-immunogenic
components selected. In such tests, the polymeric protein is administered and, after an
appropriate period of time to allow an immune response to develop against a putative
antigen, for example two weeks, a blood sample is tested to determine the level of
antibodies directed to the putative antigen, using ELISA.
Suitable animal models may also be used to test for the presence of an antigen portion
that causes antigen-specific immunosuppression or a portion that acts as an antigen
when administered to the mammalian subject. For example, the mouse model of ITP
described in Example 4 may be used. The extent and type of immune response to the
antigen is then tested following administration of the polymeric protein lacking the
antigen or putative antigen, or polymeric protein comprising the antigen or putative
antigen.
Suitably, the polymeric protein for use according to the first aspect of the invention does
not activate the classical pathway of complement, although it may be capable of binding
to C1q (Mekhaiel et al, 201 1a). Complement binding and activation can be assessed by
ELISA on wells coated with polymeric protein or control IgG or monomeric Fc as
described in Lewis M et al, Mol. Immunol 2008, 45: 818-827, and as performed in
Example 3. Wells of 96-well plates were coated overnight with 100m I protein in
carbonate buffer, pH 9. Following washing, plates are incubated with 00mI human
serum diluted 1/100 in veronal buffered saline containing 0.5mM MgCI2, 2mM CaCI2,
0.05% Tween-20, 0.1% gelatin and 0.5% BSA for 1h at room temperature. After
washing, plates are incubated with 100m I of either a 1/800 dilution of sheep anti-C1q-
HRP (Serotec) or a 1/500 dilution of biotin-conjugated anti-C5b-9 (Quidel, Santa Clara,
CA), followed by 100m I streptavidin-HRP (Dako) diluted 1/1000 in PBS-T, 0.5% BSA for
1h at room temperature. Absorbance values below 0.4 are considered negative. In a
typical assay for C1q binding, absorbance values above 0.4 are achieved when the
plates are coated with polymeric protein of above 2 mg/ml, or above 4 mg/ml or above 10
mg/ml. In a typical assay for C5b-9 deposition, indicative of complement activation,
absorbance values above 0.4 are not achieved when the plates are coated with
polymeric protein of up to 2 g/ml, up to 4 g/ml, up to 10 g/ l, up to 20 mg/ml or up to
50 mg/ml.
Suitably, the polymeric protein for use according to the first aspect of the invention is
capable of binding Protein G or Protein A with sufficient avidity to permit either substrate
to be used as a capture agent for purification of the polymeric protein. Protein G binds
in the CY2-Cy3 interdomain region and was used to purify the hexameric and monomeric
Fc proteins in the Examples. Other receptors which bind to the same region may also
bind the polymeric protein, such as TRIM21 (Mallery DL et al, 2010, Proc. Natl. Acad.
Sci. U.S.A. 107 (46): 19985-19990; McEwan WA et al 201 , BioEssays. 33: 803-809).
TRIM21 is thought to be important in removal of and immunity to viruses.
Suitably, the polymeric protein for use according to the first aspect of the invention has a
molecular weight of from about 230 to 400 kD. For example, it may have a molecular
weight of about 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390 or 400 kD. Polymers within this size range are typically easier for
mammalian cells to synthesise and assemble than molecules that have larger molecular
weights, like IgM of ~750kDa, or complex proteins like antibodies that require expression
and assembly of two different polypeptide chains (light and heavy chain). Proteins with
multiple different polypeptide chains, including antibodies, are more difficult to produce to
homogeneity during manufacture.
Suitably, the polymeric protein for use according to the first aspect of the invention has a
diameter of about 20 nm, such as from 15 to 25 nm or up to 30 nm. 20 nm particles
have been shown to be more readily delivered to lymphatics than 45 and 100 nm
nanoparticles, which may be important for function and tissue penetration (Reddy et al,
2007). Smaller particles are also more easily manufactured by recombinant expression
technology.
As a consequence of the molecular size and diameter, the polymeric protein typically has
a good degree of tissue penetration, which may assist its therapeutic effect (Vollmers &
Brandlein, 2006). In comparison with small molecules or monomers, polymers will
naturally show a slower penetration over time, but even intact IgM molecules (750 kDa)
reach implanted tumours in mice and primary tumours and metastases in patients after
'.v. or i.p. administration (Vollmers et al, 1998a,b, Oncol Rep 5: 549-552; Oncol Rep
5:35-40). It can be demonstrated by standard immunohistochemical techniques that
pentameric IgM, when injected i.p. can reach and shrink subcutaneously transplanted
tumours on the backs of animals (all the Vollmers refs, supra). This shows that
molecules as large as 750 kDa are able to leave the peritoneal cavity, enter the
circulation and reach implanted tumours. On their way, the molecules have to pass
several endothelial barriers of lymphatic and blood vessels before they reach their
targets, Jain et al, 2001 J. Control. Release 74: 7-25. Taken together, it is predicted that
polymeric proteins for use according to the invention, including the exemplified
hexameric Fc protein, will show intermediate penetrance times (hours) between the
larger IgM (days) and smaller IgG (minutes). For conditions like ITP requiring a biological
activity, an intermediate penetration and accumulation may be an advantage over
treatments that use antibodies as carriers where fast penetrance is preferred.
Suitably, the polymeric protein for use according to the first aspect of the invention has a
spatial orientation with respect to the cell surface which is cis (each Fc is in the same
plane parallel to the cell surface and the long axis of each Fc is perpendicular to the cell
surface), rather than trans. The cis orientation permits the ligation of multiple receptors
on the same cell, because the receptor binding portions are all closely apposed to the
same cell. In contrast, a trans orientation in which the Fes are in a plane perpendicular
to the cell surface might result in cross-linking of two cells by the same protein. In the cis
orientation, the biological effects of receptor binding are focussed on a particular cell,
and therefore are more likely to be productive.
The polymeric proteins are intended for treatment of autoimmune or inflammatory
diseases, according to the first aspect of the invention. "Autoimmune disease" includes
any disease in which the immune system attacks the body's own tissues. "Inflammatory
disease" includes any disease characterised by a destructive inflammation which may be
recurrent or chronic and is not associated with normal tissue repair. Such diseases
particularly include "autoinflammatory diseases" in which the innate immune system
causes inflammation which may be for unknown reasons. Autoinflammatory disorders
are characterized by intense episodes of inflammation that result in such symptoms as
fever, rash, or joint swelling. These diseases also carry the risk of amyloidosis, a
potentially fatal build up of a blood protein in vital organs.
Suitable autoimmune or inflammatory diseases for treatment include those that are
treatable with intravenous immunoglobulin (IVIG). These may be diseases which are
currently routinely treated with IVIG or in which IVIG has been found to be clinically
useful, such as autoimmune cytopenias, Guillain-Barre syndrome, myasthenia gravis,
anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis (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 e al, Lancet 337, 1 37 (1991);
LeHoang P et al, Ocul. Immunol. Inflamm. 8, 49 (2000). 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. In addition, other medical conditions
having an inflammatory component are included, 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.
Alternatively, 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.
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.
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.
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.
An exemplary disease for treatment in idiopathic thrombocytopenic purpura (ITP).
Polymeric proteins for use in a given species typically include Fc portions from the IgG of
that species, and may also include tailpiece portions from the IgA or IgM of that species.
Typically the mammalian subject to be treated is a human, although other mammals and
indeed birds, amphibians and reptiles may be treated.
Polymeric proteins are typically provided as appropriately formulated therapeutic
compositions. Suitably, they are provided as injectables either as liquid solutions or
suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection
may also be prepared. The preparation may also be emulsified. In addition, if desired,
minor amounts of auxiliary substances such as wetting or emulsifying agents, pH
buffering agents may be included.
The carrier may be preferably a liquid formulation, and is preferably a buffered, isotonic,
aqueous solution. Suitably, the therapeutic composition has a pH that is physiologic, or
close to physiologic. Suitably it is of physiologic or close to physiologic osmolarity and
salinity and/or is sterile and endotoxin free. It may contain sodium chloride and/or
sodium acetate. Pharmaceutically acceptable carriers may also include excipients, such
as diluents, and the like, and additives, such as stabilizing agents, preservatives,
solubilizing agents, and the like. As used herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of US or EU or other government or listed in the
U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans.
Pharmaceutical compositions may be formulated for any appropriate manner of
administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal,
nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein
includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular,
spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and
intraperitoneal injection, as well as any similar injection or infusion technique. Forms
suitable for oral use include, for example, tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups
or elixirs. Compositions provided herein may be formulated as a lyophilizate. Typically,
the composition is formulated for intravenous administration.
Aqueous suspensions contain the active ingredient(s) in admixture with excipients
suitable for the manufacture of aqueous suspensions. Such excipients include
suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and
gum acacia); and dispersing or wetting agents (e.g., naturally-occurring phosphatides
such as lecithin, condensation products of an alkylene oxide with fatty acids such as
polyoxyethylene stearate, condensation products of ethylene oxide with long chain
aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of
ethylene oxide with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with
partial esters derived from fatty acids and hexitol anhydrides such as polyethylene
sorbitan monooleate). Aqueous suspensions may also comprise one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring
agents, one or more flavoring agents, and one or more sweetening agents, such as
sucrose or saccharin.
The formulations may be for local or topical administration, such as for topical application
to the skin, wounds or mucous membranes, such as in the eye. Formulations for topical
administration typically comprise a topical vehicle combined with active agent(s), with or
without additional optional components. Suitable topical vehicles and additional
components are well known in the art, and it will be apparent that the choice of a vehicle
will depend on the particular physical form and mode of delivery. Topical vehicles include
water; organic solvents such as alcohols (e.g., ethanol or isopropyl alcohol) or glycerin;
glycols (e.g., butylene, isoprene or propylene glycol); aliphatic alcohols (e.g., lanolin);
mixtures of water and organic solvents and mixtures of organic solvents such as alcohol
and glycerin; lipid-based materials such as fatty acids, acylglycerols (including oils, such
as mineral oil, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids
and waxes; protein-based materials such as collagen and gelatin; silicone-based
materials (both non-volatile and volatile); and hydrocarbon-based materials such as
microsponges and polymer matrices.
A pharmaceutical composition may be formulated as inhaled formulations, including
sprays, mists, or aerosols. For inhalation formulations, the compounds provided herein
may be delivered via any inhalation methods known to those skilled in the art. Such
inhalation methods and devices include, but are not limited to, metered dose inhalers
with propellants such as CFC or HFA or propellants that are physiologically and
environmentally acceptable. Other suitable devices are breath operated inhalers,
multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the
subject method typically include propellants, surfactants and co-solvents and may be
filled into conventional aerosol containers that are closed by a suitable metering valve.
Inhalant compositions may comprise liquid or powdered compositions containing the
active ingredient that are suitable for nebulization and intrabronchial use, or aerosol
compositions administered via an aerosol unit dispensing metered doses. Suitable liquid
compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable
inhalant solvent, e.g., isotonic saline or bacteriostatic water. The solutions are
administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by
any other conventional means for causing or enabling the requisite dosage amount of the
liquid composition to be inhaled into the patient's lungs. Suitable formulations, wherein
the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops,
include aqueous or oily solutions of the active ingredient.
Formulations or compositions suitable for nasal administration, wherein the carrier is a
solid, include a coarse powder having a particle size, for example, in the range of 20 to
500 microns which is administered in the manner in which snuff is administered (i.e., by
rapid inhalation through the nasal passage from a container of the powder held close up
to the nose). Suitable powder compositions include, by way of illustration, powdered
preparations of the active ingredient thoroughly intermixed with lactose or other inert
powders acceptable for intrabronchial administration. The powder compositions can be
administered via an aerosol dispenser or encased in a breakable capsule which may be
inserted by the patient into a device that punctures the capsule and blows the powder out
in a steady stream suitable for inhalation.
The actual dosage amount of a composition of the administered to a mammalian subject
can be determined by physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or concurrent therapeutic
interventions, idiopathy of the patient and the route of administration. The practitioner
responsible for administration will, in any event, determine the concentration of active
ingredient(s) in a composition and appropriate dose(s) for the individual subject.
Pharmaceutical compositions may comprise, for example, at least about 0.1% of an
active compound. In other embodiments, an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about 25% to about 60%,
for example, and any range derivable therein. In other non-limiting examples, a dose
may also comprise from about 1 milligram/kg/body weight, about 5 milligram/kg/body
weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100
milligram/kg/body weight per administration, and any range derivable therein. The
effective polymeric protein dose is generally from about 1% to about 20% of the effective
IVIG dose. The effective IVIG dose may generally be in the range of about 100 mg/Kg to
about 2 grams/Kg, depending on the condition to be treated.
Suitability of polymeric proteins and therapeutic compositions may be tested in animal
models, prior to administration to human patients. For an animal model to be suitable, it
is important that the Fc receptors in the animal are capable of binding to the Fc receptor
binding portions of the polymeric protein. It is known that human immunoglobulins can
bind to mouse Fc receptors. For example human IgM binds to the mouse FcM-receptor.
Pleass RJ, 2009 Parasite Immunology 31: 529-538 reports which Fc receptors can bind
which antibodies from which species. Nevertheless, where the polymeric protein
comprises Fc binding portions derived from human immunoglobulin heavy chain
sequences, it may be advantageous to use transgenic mice which express human Fc
receptors. A suitable transgenic mouse expresses the human FcyRI receptor (CD64)
which binds to human lgG1 and lgG3 (Heijnen IA et al, J Clin Invest. 1996 Jan
15;97(2):331-8). Transgenic mice expressing low affinity FcRs are also available, such
as FcyRI IA (CD32) (McKenzie SE 2002, Blood Rev 16:3-5).
Suitable mouse models are the mouse model of ITP described in Example 4. Other
suitable models include collagen-induced arthritis in mice (Jain et al, 2012) or non-obese
diabetes (NOD) mouse model (Inoue, Y. et al. (2007) J. Immunol. 179, 764-774).
According to a second and third aspect of the invention, a method for treatment of a
mammalian subject for an autoimmune or inflammatory disease is provided.
According to the second aspect, the subject is treated by administering a polymeric
protein comprising five, six or seven polypeptide monomer units; wherein each
polypeptide monomer unit consists of an Fc receptor binding portion consisting of two
immunoglobulin G heavy chain constant regions; and, optionally, a polypeptide linker
linking the two immunoglobulin G heavy chain constant regions as a single chain Fc;
wherein 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.
According to the third aspect, the subject is treated by administering a polymeric protein
consisting of five, six or seven polypeptide monomer units; wherein each polypeptide
monomer unit consists of an Fc receptor binding portion and a tailpiece region; wherein
the Fc receptor binding portion consists of two immunoglobulin G heavy chain constant
regions; and, optionally, a polypeptide linker linking the two immunoglobulin G heavy
chain constant regions as a single chain Fc; wherein each modified human
immunoglobulin G heavy chain constant region comprises a cysteine residue which is
linked via a disulfide bond to a cysteine residue of a modified human immunoglobulin G
heavy chain constant region of an adjacent polypeptide monomer unit; and wherein the
tailpiece region is fused to each of the two modified human immunoglobulin G heavy
chain constant regions of the polypeptide monomer unit, and facilitates the assembly of
the monomer units into a polymer.
For either second or third aspects, the term "immunoglobulin G heavy chain constant
region" is as described in relation to the first aspect of the invention. The
immunoglobulin G heavy chain constant regions typically associate as monomer units by
disulphide linkages, such as occurs in native antibodies. Alternatively, the two constant
regions may be produced as a single amino acid chain with an intervening linker region,
i.e. as a single chain Fc (scFc).
The polymeric protein for use according to the second aspect of the invention is formed
by virtue of each immunoglobulin G heavy chain constant region comprising 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. A tailpiece is not
included. In contrast, the polymeric protein for use according to the third aspect of the
invention includes a tailpiece, which facilitates the assembly of the monomer units into a
polymer. The tailpiece is as described in relation to the first aspect of the invention. A
short linker sequence may be provided between the tailpiece region and immunoglobulin
heavy chain constant region.
In relation to the second and third aspects, when the monomer units have assembled
into a polymer, the Fc receptor binding portions are arranged in a polymeric structure
which is spatially orientated to allow each Fc receptor binding portion to bind to an Fc
receptor. The Fc receptor binding portion of each monomer unit is as described in
relation to the first aspect of the invention. Where the Fc receptor binding portion
comprises immunoglobulin heavy chain constant regions of a human IgG isotype or
variants thereof, it will typically bind to human Fcy-receptors (FcyRI, FcyRII and FcyRIII)
and/or human FcRL5 and/or CD22 and/or DC-SIGN. Polymeric proteins will have
greater avidity for Fc receptors than will the control monomeric units (which do not
polymerise because they lack the necessary cysteine residue).
The immunomodulatory properties of the polymeric protein for use according to the
second or third aspects of the invention arise from interactions between the Fc receptor
binding portions and Fc receptors and/or other receptors and components of the immune
system which interact with Fc portions. In particular, no further immunomodulatory
portion is required; and no antigen portion that causes antigen-specific
immunosuppression when administered to the mammalian subject is required. Typically,
there is no portion that acts as an antigen when administered to the mammalian subject.
Other features of the polymeric protein for use according to the second or third aspects
are described in relation to the first aspect of the invention. Typically, the polymeric
protein does activate the classical pathway of complement, although it may bind to C1q;
typically it can bind protein G or protein A; typically it has a molecular weight of from
about 230 to 400 kDa; typically it has a diameter of about 20 nm; typically it has a good
degree of tissue penetration; and typically it has a spatial orientation with respect to the
cell surface which is cis.
Polymeric proteins for use according to the second or third aspects of the invention are
intended for treatment of autoimmune or inflammatory diseases, as described in relation
to the first aspect of the invention.
A fourth aspect of the invention provides a polymeric protein comprising five, six or seven
polypeptide monomer units; wherein each polypeptide monomer unit comprises an Fc
receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
wherein 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; wherein the
polymeric protein does not comprise a further immunomodulatory portion; or an antigen
portion that causes antigen-specific immunosuppression when administered to a
mammalian subject; wherein each polypeptide monomer unit does not comprise a
tailpiece region fused to each of the two immunoglobulin G heavy chain constant
regions.
The polymeric protein of the fourth aspect is as described in relation to the polymeric
protein for use according to the first aspect, with the exception that it expressly excluded
that each polypeptide monomer unit comprises a tailpiece region fused to each of the
two immunoglobulin G heavy chain constant regions. Typically, none of the monomer
units comprise a tailpiece region. The polymeric protein does not comprise a tailpiece
region.
A fifth aspect of the invention provides a polymeric protein consisting of five, six or seven
polypeptide monomer units; wherein each polypeptide monomer unit consists of an Fc
receptor binding portion consisting of two immunoglobulin G heavy chain constant
regions; and, optionally, a polypeptide linker linking the two immunoglobulin G heavy
chain constant regions as a single chain Fc; and wherein 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.
The polymeric protein of the fifth aspect is as described in relation to the polymeric
protein for use according to the third aspect of the invention.
A sixth aspect of the invention provides a nucleic acid molecule comprising a coding
portion encoding a polypeptide monomer unit of a polymeric protein as defined according
to the fourth or fifth aspect of the invention. Nucleic acid molecules which encode
polypeptide monomer units of a polymeric protein as described in relation to the first,
second or third aspects of the invention.
Conventional recombinant DNA methodologies may be exploited for generating the
polymeric proteins, as described, for example in (Sambrook et al, "Molecular cloning: A
laboratory manual", 2001 , 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY). The monomer unit constructs preferably are generated at the DNA level,
and the resulting DNAs integrated into expression vectors, and expressed to produce the
monomer units which assemble to form the polymeric protein.
The nucleic acid molecule of the sixth aspect of the invention comprises a coding portion
which comprises, in a 5' to 3' direction, a coding sequence for an immunoglobulin G
heavy chain constant region or a single chain Fc (scFc). In appropriate embodiments,
the latter is fused in frame with a coding sequence for a tailpiece region. DNA encoding
the coding sequences may be in its genomic configuration or its cDNA configuration. It
will be appreciated that further coding sequences may be provided between the heavy
chain and tailpiece coding sequences, to allow these components to be separated from
each other in the expressed protein by linker sequences. Nucleic acids encoding linker
sequences may be included, for example, to allow the inclusion of useful restriction sites
and/or to allow the heavy chain region and tailpiece coding regions to be transcribed in
frame. Suitable coding regions can be amplified by PCR and manipulated using
standard techniques (Sambrook et al, supra). Mutations compared to native nucleic acid
sequences can be made by SOEing PCR or site directed mutagenesis.
Suitably, the coding portion of the nucleic acid molecule encodes a signal peptide, which
is contiguous with the polypeptide monomer unit. This facilitates isolation of the
expressed monomer units from a host cell. The nucleic acid molecule will therefore
comprise a coding portion which comprises, in a 5' to 3' direction, a signal sequence
fused in frame with the coding sequence of the monomer unit.
The portion of the DNA encoding the signal sequence preferably encodes a peptide
segment which directs the secretion of the monomer unit and thereafter is cleaved away
from the remainder of the monomer unit. The signal sequence is a polynucleotide which
encodes an amino acid sequence which initiates transport of a protein across the
membrane of the endoplasmic reticulum. Signal sequences which are useful include
antibody light chain signal sequences, e. g., antibody 14.18 (Gillies et al. (1989) J. OF
IMMUNOL. METH., 125: 191), antibody heavy chain signal sequences, e. g., the
MOPC141 antibody heavy chain signal sequence (Sakano et al.
(1980) NATURE 286: 5774), and any other signal sequences which are known in the art
(see, for example, Watson (1984) NUCLEIC ACIDS RESEARCH 12: 5145).
Further aspects of the invention are an expression vector comprising the nucleic acid
molecule of the sixth aspect of the invention; a host cell comprising the expression
vector; and a therapeutic composition comprising the polymeric protein of the fourth or
fifth aspect of the invention. Therapeutic compositions are as described in relation to the
polymeric proteins for use according to the first aspect of the invention.
Nucleic acid molecules as described herein may generally be part of expression vectors.
As used herein, the term "vector" is understood to mean any nucleic acid comprising a
nucleotide sequence competent to be incorporated into a host cell and to be recombined
with and integrated into the host cell genome, or to replicate autonomously as an
episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA
vectors, viral vectors and the like. Non-limiting examples of a viral vector include a
retrovirus, an adenovirus and an adeno-associated virus. As used herein, the term
"gene expression" or "expression" of monomer unit, is understood to mean the
transcription of a DNA sequence, translation of the mRNA transcript, and optionally also
secretion of a monomer unit.
Typically, host cells are provided for expression of the expression vector. The cell can
be a mammalian, avian, insect, reptilian, bacterial, plant or fungal cell. Examples of
mammalian cells include, but are not limited to, human, rabbit, chicken, rodent (e.g.
mouse, rat) cells. Typical mammalian cells include a myeloma cell, a Sp2/0 cell, a CHO
cell, L cell, COS cell, fibroblast, MDCK cell, HT29 cell, HEK cells, or a T84 cell. A
preferred host cell is CHO-K1. Expression vectors may be introduced into host cells
using standard techniques, including calcium phosphate transfection, nuclear
microinjection, DEAE-dextran transfection, bacterial protoplast fusion and
electroporation.
Polymeric proteins may be prepared by methods including (1) preparing a vector
comprising the nucleic acid molecule encoding the monomer unit; (2) transfecting a host
cell with the vector; (3) culturing the host cell to provide expression; and (4) recovering
the polymeric protein.
The polymeric protein may be recovered as products of different molecular sizes.
Typically, the desired polymeric protein comprising five, six or seven monomer units
accounts for at least 40% of the total protein composed of monomer units, by weight.
Suitably, it accounts for at least 50%, at least 60%, at least 70%, at least 80% or at least
90% of the total protein composed of monomer units, by weight. The high proportion of
monomer units that can be recovered as the desired polymeric protein contributes to
production efficiency.
If the polymeric protein is secreted by the host cell it can conveniently be recovered by
affinity chromatography utilising its affinity for Fc binding agents, such as Protein A or
Protein G, suitably Protein-G HiTrap (GE healthcare) columns. Proteins may be eluted
from such columns by low pH into neutral buffer. Dialysis may subsequently be
performed for buffer exchange. If the host cell does not secrete the polymeric protein, it
may be recovered by lysing the cells followed by affinity chromatography.
The present invention will be further illustrated in the following examples, without any
limitation thereto.
Example 1: Production of a recombinant polymeric Fc protein
DNA constructs were prepared as follows. A commercially available pFUSE-hlgG1-Fc2
expression vector was obtained from InvivoGen, sourced via Autogen Bioclear, Wiltshire,
UK. The expression vector comprises a coding sequence for a signal sequence from IL2
and, downstream of that, a coding sequence for the Fc portion from human lgG1. To
generate a polymeric protein, two changes to the coding sequence of the human lgG1
Fc-portion were made. The 18 amino-acid tailpiece from IgM was sub-cloned onto the
C-terminus of the Fc portion, and an additional mutation was made in the Cv3 domain to
convert residues 309 and 310 (EU numbering throughout) to cysteine and leucine
respectively.
In order to insert the IgM tailpiece sequence into the commercially available vector,
primers were designed which would, when annealed together, form a double stranded
sequence with overhanging bases encoding a Nhe1 restriction site to allow subcloning
C-terminal to the Fc. In order to maintain the reading frame of the protein encoded by
the plasmid, remove an existing stop codon, and allow for convenient restriction sites, an
extra DNA base was inserted. The IgM tailpiece sequence is preceded by a short 5'
linker which encodes for four amino acids Leu-Val-Leu-Gly; the linker does not affect the
function of the IgM tailpiece.
Primers 1 and 2 (SEQ ID No. 1 and 2) were annealed together via a temperature
gradient to form the double stranded IgM-tailpiece containing Nhe1 insert.
The pFUSE vector was then digested with the restriction enzyme Nhel, and the IgM
tailpiece insert above ligated to create an intermediary plasmid. In order to allow the IgM
tailpiece to be translated after the Fc region, the stop codon present was mutated in a
subsequent step, via site directed mutagenesis, utilising the Quick Change II Kit
(Stratagene, La Jolla, CA, USA). Primers 3 and 4 (SEQ ID No. 3 and 4) were designed
to remove this stop codon and create an Avrll restriction enzyme site between the Fc
region and the IgM tailpiece. This created the plasmid termed pFUSE-hlgG1-Fc-TP.
In human IgM, a cysteine at position 309 is involved in forming a disulphide bridge
between two monomers of IgM within the pentamer. In order to better mimic the protein
sequence of human IgM, primers 5 & 6 (SEQ ID No. 5 and 6) were designed to introduce
a cysteine residue at position 309, again by site-directed mutagenesis, as before. Upon
alignment of the nucleotide sequence encoding the protein sequence of human IgM with
that of human lgG1-Fc, it was decided to, in addition, replace the neighbouring histidine
residue at position 310 with a neutral leucine residue. The final plasmid incorporating
both mutations was named pFUSE-hlgG1-Fc-TP-LH309/310CL.
Control plasmids encoding monomers of human lgG1 Fc lacking the 309/310 mutations
and the tailpiece were also prepared.
The nucleic acid coding sequence for the monomeric units which assemble into polymers
is SEQ ID No. 7.
The coding sequence has the following regions:
1-60 coding sequence for IL2 signal peptide
61-753 coding sequence for Fc-region of human lgG1
325-330 cys-309 and leu-310 mutations
754-810 coding seqeunce for IgM tailpiece (including linker region and stop codon)
The amino acid sequence of the monomeric units which assemble into polymers is SEQ
ID No. 8.
The amino acid sequence has the following regions:
1-20 IL2 signal peptide
21-247 Fc-region of human lgG1
109-1 0 cys-309 and leu-310 mutations
248-251 Four amino acid linker
252-269 IgM tailpiece
During expression, the IL2 signal peptide is cleaved off and so the final protein product
has 249 amino acids.
Polymeric Fc proteins and control monomeric Fc proteins were prepared as follows.
CHO-K1 cells (European Collection of Cell Cultures) were transfected by electroporation
with plasmids and positive clones selected. The cells were grown in DMEM complete
media supplemented with 10% ultra-low bovine IgG FCS, 100 lU/ml penicillin, and
OO gml 1 streptomycin (PAA) at 37°C/5%C0 2. Stable transfectants were selected in
medium containing 400 g l 1 of Zeocin (Invivogen). Clones secreting Fc-fusion
proteins were detected by sandwich enzyme-linked immunosorbent assay (ELISA) using
goat anti-human IgG-Fc (Sigma-Aldrich: A0170). From large-scale cultures in DMEM
supplemented with ultralow IgG containing FBS (Gibco), Fc-fusion proteins were purified
on Protein-G-Sepharose (GE Healthcare, Little Chalfont, Bucks, UK). Eluted fractions
from the affinity purification were pooled and separated by size-exclusion
chromatography on a high-performance Superdex-200 10/300GL column using an
AKTAFPLC (GE Healthcare). Eluted fractions were compared against known high MW gel
filtration standards (Biorad). The integrity of the proteins was verified by SDS-PAGE on
native 6% Tris-glycine gels against SeeBlue2 pre-stained molecular weight markers
(Novex-lnvitrogen).
Polymeric proteins were expressed as complexes of about 312 kD and about 100 kD,
consistent with expression as hexameric and dimeric entities. The proportion was about
90% hexameric versus 10% dimeric (w/w) according to size-exclusion chromatography,
as illustrated in Figure 1B.
Example 2: Structural characterisation of a recombinant polymeric Fc protein
Hexameric-Fc were imaged by tapping mode atomic force microscopy (AFM) under
solution. Hexameric-Fc form a highly uniform population of well defined structures. The
obtained AFM images showed the complex to be cylindrical in structure (18±2nm (n=51)
in diameter, 5.7±0.4nm (n=54) in height). Representative images are shown in Figure
1A. The obtained images are also consistent with dimensions predicted from in silico
modelling analysis, also illustrated in Figure 1A.
AFM was performed as follows, and as described in Mekhaiel et al, 201 1a. Stock
solutions of hlgG1-Fc-LH309/310CL-TP in IxHBSS buffer were diluted to 10 gml' 1 in
0.2xHBSS buffer and then directly applied to a freshly cleaved fragment of muscovite
mica. After incubating for 20 minutes, the sample was rinsed extensively with 0.2xHBSS
buffer to remove unadsorbed molecules. The samples were always under solution
during transport to and imaging within the AFM. Imagaing was performed in the tapping
mode with a Nanoscope Ilia Multimode AFM (Veeco, Santa Barbara, CA) using silicon
nitride cantilevers (NP-S, Veeco Probes, Santa Barbara, CA) with a spring constant of
0.32N/m in 0.5xHBSS buffer. The typical scan rate and initial oscillation amplitude were
3Hz and 20nm, respectively, and the applied force was minimized to ~0.1nN. The
piezoscanner (9mih , D scanner, Veeco) was calibrated using mica and gold ruling. All
images reported were reproducible with different tips and different fast-scan directions.
All lateral dimensions were determined from the full-width at half height.
Molecular (in silico) modelling was performed as follows, and as described in Mekhaiel et
al, 201 1a. The monomer was constructed from the crystal structure of the hlgG1 Fcdomain,
using a random chain structure for both the hinge and tailpiece regions. Each
monomer was energy minimized before being assembled into the hexameric complex.
All energy calculations were performed using VMD/NAMD using the CHARMM27 force
field. NAMD was developed by the Theoretical Biophysics Group in the Beckman
Institute for Advanced Science and Technology at the University of Illinois at Urbana-
Champaign. To construct the hexamer, two experimental observations were taken into
account: first, the stoichiometry of the complex is finite (that is, oligomerization does not
proceed indefinitely), and second, oligomerization required the Cys309 mutation. The
former observation suggests that there is some physical reason preventing further
assembly, which we reasoned was owing to the complex being ring-shaped, similar to
those seen with natural IgM complexes. The latter observation suggests that adjacent
monomers are linked through these C309 residues. For these linkages to be possible in
a ring-shaped complex, these residues must all lie within a common plane, as is the case
with the homologous cysteine residues in the IgM pentamer. With these Fc-fusions,
there are two types of oligomers that can satisfy these criteria: star-shaped, in which the
C309 plane is the same as the Fc-plane, and barrel-shaped, where the C309 plane is
perpendicular to the Fc-plane. Both models were examined to determine which produced
a structure with lower energy, subject to one additional criterion. Since these Fc-fusions
have not evolved to interact with each other but rather link by virtue of the mutated C309
residues, we assumed that the monomers do not undergo significant structural changes
to form these bonds, and so assigned an energetic penalty to any such changes. This
was achieved by constraining the backbone atoms within a harmonic well (spring
constant, 1 kcal/mol/A2) centered cn the positions of the energy-minimized monomeric
structures. By monitoring the contribution of this constraining potential to the total
minimized energy, the extent to which each monomer changed conformation from the
monomeric form could be evaluated. Without this constraint, the star-shaped and barrelshaped
structures, evaluated at a range of initial C309-C309 distances, were of a similar
minimized energy. However with the constraint, at all distances, it was clear that the
barrel-shaped structure required significantly fewer structural changes than the starshaped
model to form the C309-C309 disulfide bridge. Thus, without the constraint, the
barrel-shaped structure achieves a similar minimized energy as the star-shaped
structure, but requiring fewer changes to the monomeric form. We therefore favor a
barrel-shaped structure for the hexamer. The final model was constructed at the closest
initial C309-C309 distance at which there was the smallest degree of structural changes
to the monomeric structure. These calculations were performed without any of the
tailpieces connected to other monomers. With this minimized model, the tailpieces were
connected to other, randomly selected, monomers within the complex, and then the
entire complex was solvated with TIP3 water, minimized, and finally equilibrated, as
judged by the root-mean-squared-deviation of the protein backbone.
Example 3: Interaction of a polymeric Fc protein with components of the immune
system
1. Interactions with Fc-receptors
Hexameric Fc bound to all of the Fc receptors tested, namely human FcyRI, FcyRIIA and
FcyRIIB, and mouse FcyRI and FcyRIIB. The hexameric-Fc bound with higher affinity (in
the nanomolar range) to the low affinity human FcyRs (FcyRIIA 131 and FcyRIIB) than
dimers or monomers that bind to the same receptors in the micromolar range. This
confirms that higher order polymers do have improved binding kinetics to receptors
known to be involved in protecting from ITP. In SPR analysis, Hexameric-Fc bound to
the human FcyRI with a KA (1/M) of 1.6x1010. Dimers bound with a KA (1/M) of 6.4x1 09.
Binding of hexameric or monomeric Fc to Fc receptors by ELISA is shown in Figure 2.
Another receptor that may play a role in ITP is FcyRIII (Park-Min et al, 2007).
Hexameric-Fc can be expected to bind human FcyRIII because the binding site for
FcyRIII on IgG overlaps with that of FcyRIIB and FcyRI (Sondermann et al, 2001). The
IgG lower hinge region plays a key role in the interaction with FcyRI and FcyRI I , and it
seems probable that all of the FcyRs share a common mode of interaction with IgG
(Woof & Burton, 2004). Indeed, it has been possible to produce model complexes
between IgG Fc and FcyRI and FcyRI I based on the FCYRI II—IgG Fc crystal structure and
thereby rationalize particular binding characteristics (Sondermann et al, 2001). The
interaction of FcyRIII and hexameric-Fc complex was modelled in silico using the crystal
structure of FcyRIII. The model shows that the binding site in the hexamer for FcyRIII is
most likely exposed, as it clearly is for the other FcyRs. We therefore predict that FcyRIII
would bind to the hexameric-Fc.
Binding of Fc proteins to Fc receptors was determined by enzyme linked immunosorbent
assays (ELISA) as described below and in Mekhaiel et al, 201 1a. Microtiter wells (Nunc)
were coated with titrated amounts of the Fc proteins (50-0.3 nM) in PBS or carbonate
buffer pH9 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 7.4 before addition of GST or HIS-fused hFcyRI,
hFcyRIIA, hFcyRIIB, mFcyRI or mFcyRIIB diluted in 4% skimmed milk PBS/0.005%
Tween 20 (PBS/T) pH 7.4 and added to the wells. After incubation for 2 h at RT and
washing as above, an HRP-conjugated polyclonal anti-GST from goat (1:8000; GE
Healthcare) was added and incubated for 1 h at RT. Wells were washed as above, 100
m I of the substrate TMB (Calbiochem) was added to each well and incubated for 45
minutes before 100 m I of 0.25 M HCI was added. The absorbance was measured at
450 nm using a Sunrise TECAN spectrophotometer (TECAN, Maennedorf, Switzerland).
Binding to human FCYRI was also analysed by surface plasmon resonance analysis,
exactly as described below in relation to FcRn.
2. Interactions with glycan receptors known to play a role in controlling autoimmunity
Human DC-SIGN and its homologue in mice (SIGN-R1) have been shown to be involved
in controlling ITP by binding the terminal sialic acid found on the glycan attached at N297
of the Fc of IgG (Anthony et al, 201 1; Samuelsson et al, 2001). Sialylation of hexameric-
Fc protein was demonstrated as follows. Reduced proteins were run on 4-12% Bis-Tris
gels and transferred to PVDF membranes and blocked with 1% Roche blocking reagent
as described previously (Samuelsson et al, 2001). Membranes were then incubated with
a 1/200 dilution of biotinylated Sambucus nigra bark lectin (Vector laboratories) and
developed with streptavidin-HRP (Serotec). In silico modelling predicts that these
terminal sialic acids found on hexameric-Fc proteins would be available to bind DC-SIGN
or SIGN-R1 (Figure 1).
3. Interactions with newly described receptors that may play a role in controlling
autoimmune disease
Human B cell inhibitory receptors are important in controlling autoimmune disease
(Pritchard & Smith, 2003). Two inhibitory receptors for IgG have been described on the
surface of human B cells: FcyRllb (Daeron et al, 1995) and more recently FcRL5 (Wilson
et al, 2012). Although it is possible that FcRL5 is redundant with FcyRllb in human B
cells, the potential for simultaneous recruitment of SHIP-1 by FcyRllb and SHP-1 to
FcRL5 provides a substantial barrier to recurrent activation of B cells. Intriguingly,
FcRL5 on innate B cells is also targeted by Poxvirus MHC Class l-like immunoevasins,
suggesting an important role for FcRL5 in regulating immunity (Campbell et al, 2010).
FcRL5 only binds complexed IgG and not monomeric IgG and therefore IVIG would be
less likely to bind this receptor with high avidity, although the polymeric fraction of IVIG
might bind with high avidity. Taken together, FcyRllb and FcRL5 may limit B cell
activation against chronic pathogens or self-reactive antigen, and approaches that have
the potential to target both receptors may prove beneficial in therapies aimed at
controlling B cell activation.
We therefore investigated the binding of hexameric-lgG1-Fc to human CD19+ B cells
(Figure 3). As a first step to evaluate the interaction of hexameric lgG1-Fc with immune
cells involved in controlling autoimmunity, we investigated the ability of hexameric lgG1-
Fc to bind human B cells by FACS analysis. We show that hexameric lgG1-Fc bound to
CD19+ B cells purified from the peripheral circulation of healthy human volunteers. Flow
cytometry was performed as described in Mekhaiel et al, 20 1a. Human leucocytes
were purified from whole blood by centrifugation on Polymorphoprep gradients according
to manufacturer's instructions. 1 x 105 cells were incubated with 200 m I FACs buffer
(phosphate-buffered saline, 0.2% bovine serum albumin, 5% goat serum) containing 50
g of hexa-Fc or buffer only for 1h at room temperature. Cells were washed twice with
FACs buffer and incubated for 1h at 4°C with 1/500 dilution of F(ab')2 goat anti-hlgG-Fcphycoerythrin
(PE) and goat anti-hCD19-fluorescein isothiocyanate (FITC)-conjugated
Abs (Southern Biotechnology) in 200 m I FACs buffer. After washing with FACs buffer,
cells were analyzed on a FACScan (BD Biosciences). Data acquisition was conducted
with CELLQuest software (BD Biosciences) and the analysis performed with FlowJo
version 9.1. CD19+ B lymphocytes were gated taking into account their forward and side
scatter profiles, and binding of Fc protein was detected with the PE-labelled secondary
antibody. No binding of the PE-labelled secondary antibody was detected in the
absence of the Fc protein. That hexamer-Fc was binding FcRL5 on the surface of human
B cells was demonstrated by prior incubation of cells with FcRL5 specific blocking
monoclonal antibody 509F6, which ablated binding of the hexameric Fc protein.
4. Binding to the neonatal FcRn
We investigated the ability of hexameric-Fc to interact with neonatal FcRn, responsible
for maintaining the long half-life of Abs in the circulation (Mekhaiel et al, 201 1a).
Although FcRn does not play a dominant role in ITP, it may play a role in hexameric-Fc
mediated control of more chronic autoimmune disease, where a long half-life for the
therapy is important. Although hexameric-Fc was unable to bind human FcRn it did bind
with nM affinity to mouse FcRn (0.1-10 nM at pH6) in line with previous observation
(Andersen et al, 2010). This suggests that minor modifications to the existing construct
e.g. reversion of Leu310 to His310 may reinstate binding to human FcRn.
Binding of Fc proteins to FcRn was determined by surface plasmon resonance analysis
and as described below and in Mekhaiel et al, 201 1a. Surface Plasmon Resonance
(SPR) analyses were carried out using a Biacore 3000 instrument (GE Healthcare).
Flow cells of CM5 sensor chips were coupled with recombinant forms of soluble human
or mouse FcRn (hFcRn or mFcRn; ~ 2200-3000 RU) using amine coupling chemistry as
described in the protocol provided by the manufacturer. The coupling was performed by
injecting 2 gm 1 of the protein in 10 mM sodium acetate pH 5.0 (GE healthcare).
Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCI, 0.005% Tween 20) at pH 6.0,
or HBS-EP buffer (0.01 M HEPES, 0.15 M NaCI, 3 mM EDTA, 0.005% surfactant P20)
was used as running buffer and dilution buffer. 100 nM of each of the Fc-fusion proteins
were injected over immobilized receptor at pH 6.0 or pH 7.4, with a flow rate of 20 m I/min
at 25 °C. Regeneration of the surfaces was done using injections of HBS-EP buffer at pH
7.4 (GE Healthcare). In all experiments, data were zero adjusted and the reference cell
subtracted. Data were evaluated using the BIAevaluation 4.1 software (GE Healthcare).
5. Binding to complement
Most Fc-fusions studied to date do not bind C1q and are unable to activate the ensuing
complement cascade (Czajkowsky et al, 2012). This inability of Fc-fusions to activate
complement likely stems from the lack of Fab residues in the fusion that contribute to
interactions of intact IgG with C1q (Gaboriaud et al, 2003). Because Hexameric-Fc lacks
Fab residues it is unlikely to be able to bind C1q with the same affinity seen with native
antibodies. Nonetheless we did investigate the ability of hexameric-Fc to bind C1q and
activate the classical pathway (Mekhaiel et al, 201 1a). Hexameric-Fc bound C1q poorly
compared to dimers, which in turn bound less well than intact monomeric IgG, a
gradation reflected in commensurate decreases in detection of the C5-9 terminal
complex. There was also a complete lack of an Arthus reaction, a type III
hypersensitivity reaction initiated by immune-complex formation at the site of inoculation,
even when these reagents were administered to mice i.p. or when administered s.c.
together with alum, observations made at the time of injection. The lack of complement
activation is generally considered a desirable property of injected therapies aimed at
treating autoimmune disease. However, C1 binding without subsequent activation of the
classical pathway may be advantageous in light of the finding that DC-SIGN, C1q, and
gC1qR form a trimolecular receptor complex on the surface of monocyte-derived
immature dendritic cells that may be important in controlling autoimmune disease
(Hosszu et al, Blood 2012; 120: 1228-1236).
The Arthus reaction involves the in situ formation of antigen/antibody complexes after the
intradermal injection of an antigen (as seen in passive immunity). If the animal/patient
was previously sensitized (has circulating antibody), an Arthus reaction occurs. Typical of
most mechanisms of the type III hypersensitivity, Arthus manifests as local vasculitis due
to deposition of IgG-based immune complexes in dermal blood vessels. Activation of
complement primarily results in cleavage of soluble anaphylotoxins C5a and C3a, which
drive recruitment of PMNs as well as local mast cell degranulation (requiring the binding
of the immune complex onto FcyRIII), resulting in an inflammatory response. Further
aggregation of immune complex-related processes ensures in the tissue vessel walls a
local fibrinoid necrosis with ischemia-aggravating superimposed thrombosis. The end
result is a localized area of redness, readily observable by eye at the site of injection that
in duration typically lasts a day or so.
Example 4: Hexameric-Fc can restore platelet counts in a clinically relevant mouse
model of ITP.
In a commonly used platelet depletion model for ITP we could show that as little as
0.035g/kg of hexameric Fc had a significant positive effect on platelet recovery by
comparison with a similar dose 0.035g/kg of IVIG.
Balb/C mice were injected i.p. with Hexa-Fc at 0.035g/kg, IVIG at 0.035g/kg or 2g/kg
(GammaGard), or PBS in a final volume of 200 m I. One hour later ITP was induced in all
mice by i.p. injection of 12.5 g lgG1 -K specific for mouse integrin b3 chain/CD61, clone
2C9.G2 (BD Pharmingen; Product #: 550541) in a 200m I volume of sterile PBS. Platelets
were enumerated at 12h, 24h, 36h and 48h time-points post treatment by flow cytometry
using an APC-conjugated anti-CD61 antibody n = 4 mice per group from 2 independent
experiments. Group means ±SD were analyzed by repeated ANOVA using Dunnett's
multiple comparison test versus the control group. Control vs hexamer: q=3.13, * P<0.05;
control vs IVIG: q=8.66,***P<0.0001 . Data are mean % platelets ±SD. Mean % platelet
counts in each group prior to depletion: hexamer group = 9.6%±1 .2, high dose IVIG
group = 9.4%±1.2, low dose IVIG group = 9.4%±1 .2, PBS group = 9.3%±1.2.
Example 5: Treating patients with ITP
Treatment protocols for ITP with the disclosed hexameric Fc protein would be utilized in
a manner tracking standard guidelines for ITP hIVIG therapy such as the Executive
Committee of the American Society of Hematology practice guideline for the diagnosis
and management of primary immune thrombocytopenic purpura. See George, J N, et al.
Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods
for the American Society of Hematology. Blood. 1996 Jul. 1; 88(1):3-40. Alternatively,
the protocols for ITP may include an initial administration phase with dosages of about
0.1 to about 0.001 times the above treatment protocol dosages. The initial low dose
phase is designed to minimize any short term pro-inflammatory effects of the Fc protein
administration while still being sufficient to induce a long term anti-inflammatory effect,
which is subsequently enhanced and maintained by the second phase standard dosing
described above. The rationale for this alternative approach is that some polymeric
proteins may have both a short term inflammatory effect as well as a long term anti¬
inflammatory effect through decreasing the expression of FcyRlla. An initial low dose (or
initial low doses) can be used to stimulate the long term anti-inflammatory effect while
minimizing the short term inflammatory effect.
The Fc protein would be administered intravenously and the effective Fc protein dose is
generally from about 1% to about 20% of the effective IVIG dose. The effective hIVIG
dose in ITP is generally in the range of about 100 mg/Kg to about 2 grams/Kg
administered every 10-21 days.
The Fc protein intravenous formulation will be substantially the same as FDA approved
hIVIG formulations but may exclude the stabilizers present in some hIVIG formulations.
Example 6: In silico modelling of Fc proteins
The ability of Fc proteins to modulate autoimmune or inflammatory diseases will depend
on their ability to interact with appropriate components of the immune system, whilst not
provoking unwanted reactions. This in turn will depend on the structure of the Fc protein.
In the case of oligomers, tertiary and quaternary structure will have an important impact
on the availability of protein surfaces and exposed moieties for binding to components of
the immune system. A uniform and defined tertiary and quaternary structure may reduce
the risk of unwanted reactions which might otherwise be provoked by minority species
within complex mixtures. A uniform and defined structure also permits easier quality
control of medicinal products.
The inventor has shown that hexameric-Fc disclosed herein has a regular and uniform
cylindrical shape (Example 2), binds to various desirable receptors and components of
the immune system (Example 3) and does not activate complement (Example 3). These
findings are all consistent with the in silico modelling of hexameric-Fc described in
Example 1.
US 2010/0239633 (University of Maryland, Baltimore; Strome) discloses IVIG
replacement compounds comprising multiple linked Fc portions. Linear or pentameric
arrangements are envisaged. Pentamerization is envisaged by the addition of the Om4
domain of Ig and the J chain (as depicted in Figs 10A-D and on page 18 preferred
embodiments of US201 0/0239633). In native IgM, the C 4 domain of the heavy chain
constant region binds to the J chain to effect pentamerization. Extensive in silico
modelling/molecular dynamics simulations were performed to determine if the structures
proposed in US201 0/0239633 are viable.
In the structures depicted in Fig 10A-D of US201 0/0239633, a m4 domain is linked via
its N-terminus to the C-terminus of a Cy3 domain (i.e. the CH3 domain of an IgG
molecule), and the Cy3 domain is linked to a Cy2 domain. Like domains of the
monomers are depicted as dimerizing together to form monomer arms of the higher
order structure. However, this presents a conformational problem for the structures.
In native immunoglobulins, the m3, Om4, Cy2 and Cy3 domains form homodimers which
are 'V'-shaped, or inverted 'V'-shaped, i.e TV. One monomer forms the 'V part of the 'V
and a like monomer forms the T' part of the 'V; or one monomer forms the '/' part of the
TV and a like monomer forms the 'V part of the TV. Cy3 or Om4 dimers are 'V'-shaped
whereas Om3 and Cy2 are TV-shaped. In a native immunoglobulin, V and TV-shaped
dimers alternate. For example, the N-termini of the 'V'-shaped Cy3 domains are
positioned to be contiguous with the C-termini of the TV-shaped Cy2. Likewise, the Ntermini
of the 'V'-shaped Om4 domains are positioned to be contiguous with the C-termini
of the TV-shaped Om3 domains.
In the monomers depicted in US201 0/0239633, the 'V'-shaped Cy3 domain is linked
directly to another 'V'-shaped m4 domain. This is a problem since the C-terminal
residues of the Cy3 are close to each other whereas the N-term residues of the C 4 are
far from each other. It is likely that a linker would be required to join the domains
together. The most likely result of this structural limitation is that, for the linker lengths
that would most likely be employed, the Om4 domains would have to be rotated by 90
degrees with respect to the Og2-Og3 domains. The Om4 domain would be under
rotational strain. The intra-monomer disulphide linkages within the Cy3s and the intramonomer
disulphide linkages within the Cp4s are in competition with each other. One or
both will be weakened. Therefore, the protein might be unstable or might not fold
properly.
To mimic the normal orientation of the g 2- g 3 domains with respect to the Ci4 domain
as is found in g 2- g 3- m4, domains of IgM would require linkers in excess of 20 amino
acids. The introduction of linkers (unspecified sequences) raises significant problems for
these structures, not least of which is the increased likelihood of unwanted
immunogenicity. There would also be a negative impact on manufacturability and
increased likelihood of proteolytic cleavage. This geometric limitation of the g 3- m4
linkage has significant consequences for the oligomer that make it fundamentally
different from the hexameric-Fc protein.
The linker sequences between the FCY and m domains are critical. However, although
the term 'linker' connotes flexibility, there would be limitations to this flexibility in the
present case. With a single point connection, such as between a Fab and an Fc region
(at the hinge), there can indeed be a great deal of rotational flexibility. The difference in
the embodiments as depicted in US2010/0239633 is that there are two places of linkage,
one between each g 3- m4 pair. So rotations of one Fc relative to the other Fc results
in a twisting together of the two linkers, which is energetically unfavourable, increasing
the risk of mis-folding or instability.
It is improbable that the structures described in US201 0/0239633 would form welldefined
oligomers (as in IgM) with their linkages only by the m4 domain. The 18 aminoacid
C-terminal tails found in all polymeric antibodies, being unstructured, cannot be
responsible for the regular pentameric and/or hexameric structure - that must come from
the presence of a structured Ig-domain(s) within the Fc. With polymerization occurring
through the structured Ig regions, the stoichiometry is set by the size and shape of the
structured regions. Interactions between random unstructured regions must necessarily
lead to a wide range of polymeric sizes. So, to determine whether the (few) intermonomer
contacts in the m4 domains observed in IgM would be sufficient to maintain
the pentameric stoichiometry, we undertook extensive molecular dynamics simulations of
just these structured regions (i.e. lacking tailpieces). During simulations, the structured
regions of m4 quickly lose their pentameric arrangement, and begin to (apparently)
clump together (non-specifically). A regular pentameric/hexameric structure is thus very
improbable. This is to be contrasted with native IgM, in which both the m4 and m3
domains contribute to the regular pentameric/hexameric structure. Although
US2010/0239633 suggests using the J-chain to facilitate polymerisation, this would not
be expected to be successful. The J-chain can stabilize immunoglobulin structures, but
cannot convert a monomer or dimer into a higher order oligomer. For example, the Jchain
can facilitate dimerisation of IgA, but it is not sufficient to form higher order
oligomers as in IgM, because the Ig domains of IgA are not suitable for higher order
polymerisation.
The most likely structure that could possibly form if polymerization could occur through
the random tails is a 3d star-shaped structure, where the tails are located within the
central corona and the Fc-regions of the monomers project out radially, in 3d. With
polymerization occurring through unstructured peptides, there can be no well-defined
stoichiometry and whatever can fit in three dimensions will occur. There is the possibility
of dimers up to probably 20-mers depending on how many linking tails can fit inside the
central sphere of interacting tails.
It is also important to note that experimentally, we have found that the addition of the 18
amino-acid tailpiece to the Fc of human lgG1 is insufficient to induce multimerization
(Mekhaiel, 201 1a). We have also shown that with constant domain swaps, the presence
of the m4 domain is unable to induce pentamer and/or hexamer formation (Ghumra et
al, 2009). Thus, we consider the possibility that the structures in US201 0/0239633 can
actually polymerize, whether by m4 domains or by the tails, as unlikely.
Even if the predictions arising from the models are wrong and the m4 domains in the
monomers are able to interact to form structures similar to IgM, the resulting structures
would be very different to the hexameric-Fc. Assuming that the m4 interact as they do
in gM (which is assumed by US201 0/0239633), the need for the m4 domains to be
rotated by 90 degrees with respect to the g2- g3 domains would cause the g2- g3
regions to (largely) 'face' each other in any oligomer which was able to form from the
monomers. That is, the side of the g2- g3 would directly face the opposing side of
the g2- g3 in the neighbouring arm. The overall structure would resemble a closed
umbrella. In contrast, in the hexameric-Fc protein, these sides form the walls of the
'barrel' and thus do not face each other, but rather face outwards. Receptors that can
easily bind to these sides in the hexameric-Fc protein might thus not bind to the
US2010/0239633 oligomers. The sugared regions in the g2- g3 Fc unit are critical to
binding by both Fc- and glycan-receptors. In the hexameric-Fc, the sugared regions face
out, so receptors that bind to the sugared regions can bind. In contrast, in the
US201 0/0239633 oligomers, the receptors for these glycans would have to get in
between the monomers, and although not impossible, this would nonetheless impact on
overall binding strengths between the oligomer and critical receptors. While a single
monomer of the US201 0/0239633 oligomer might be orientated so that a single pair of
Cv2-Cy3 domains is accessible to receptors, it is not clear whether more than one other
monomer in the oligomer could bind to a different receptor on the same surface, owing to
geometric considerations described above and to the physical size of the monomers.
Thus, it is not clear whether the US201 0/0239633 oligomer could bind to more than
one/two receptors simultaneously. Similar issues would arise from the use of alternative
polymerizing constant domains e.g. those from IgA. Thus, despite the presence of
multiple monomer units in the structures envisaged in US201 0/0239633, the binding to
critical Fc receptors and other components of the immune system might be no more avid
than is observed with monomeric or dimeric immunoglobulins, if such structures can form
at all.
This building block strategy seriously limits the interaction possibilities of the structures
described in US201 0/0239633. More problematic is that even if these molecules can be
shown to multimerize, the presence of the C i4 domain will have serious impact on the
bio-distribution and in vivo interactions possible from these chimeric molecules that are
expected to be significantly different to those seen with hexameric-Fc as described in this
application. These are summarized in detail in Table 1.
Table 1: Differences between Hexameric-Fc and structures envisaged
US2010/0239633
immunogenic they are non-immunogenic
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SEQUENCE LISTING
<110> Liverpool School of Tropical Medi
<120> Immunomodulatory proteins
<130> LIVBM/P50745PC
<160> 14
<170> Seq in
<210> 1
<211> 64
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 1
<400> 1
ctaggacccc ccctgtacaa cgtgtccctg gtcatgtccg acacagctgg cacctgctac 60
tgag 64
<210> 2
<211> 64
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 2
<400> 2
ctagctcagt agcaggtgcc agctgtgtcg gacatgacca gggacacgtt gtacaggggg 60
ggtc
<210> 3
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 3
<400> 3
ctgtctccgg gtaaattagt cctaggaccc cccctg 36
<210> 4
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 4
<400> 4
12 052561
cagggggggt cctaggacta atttacccgg agacag 36
210> 5
211> 48
212> DNA
213> Artificial Sequenc
220>
223> primer 5
<400> 5
gtggtcagcg tcctcaccgt ctgcctccag gactggctga atggcaag 48
<210> 6
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 6
<400> 6
cttgccattc agccagtcct ggaggcagac ggtgaggacg ctgaccac 48
<210> 7
<211> 810
<212> DNA
<213> Artificial Sequence
<220>
<223> Fc protein monomer unit coding sequence
<400> 7
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 120
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 180
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 240
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 300
cgtgtggtca gcgtcctcac cgtctgcctc caggactggc tgaatggcaa ggagtacaag 360
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 420
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 480
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 540
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 600
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 660
aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 720
ctctccctgt ctccgggtaa attagtccta ggaccccccc tgtacaacgt gtccctggtc 780
atgtccgaca cagctggcac ctgctactga 810
<210> 8
<211> 269
<212> PRT
<213> Artificial Sequence
<220>
<223> Fc protein monomer unit protein sequence
<400> 8
Met Tyr Arg Met Gin Leu Leu Ser Cys lie Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
20 25 30
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
35 40 45
Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
50 55 60
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
65 70 75 80
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr
85 90 95
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Cys Leu Gin Asp
100 105 110
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
115 120 125
Pro Ala Pro lie Glu Lys Thr e Ser Lys Ala Lys Gly Gin Pro Arg
130 135 140
Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
145 150 155 160
Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
165 170 175
e Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys
180 185 190
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
195 200 205
Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser
210 215 220
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser
225 230 235 240
Leu Ser Leu Ser Pro Gly Lys Leu Val Leu Gly Pro Pro Leu Tyr Asn
245 250 255
Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr
260 265
<210> 9
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> g linker
<220>
<221> g linker
<400> 9
Leu Val Leu Gly
1
<210> 10
<211> 18
<212> PRT
<213> Homo Sapiens
<220>
<221> Tailpiece region of human IgM
<400> 10
Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr
1 5 10 15
Cys Tyr
<210> 11
<211> 18
<212> PRT
<213> Homo sapiens
<220>
<221> Modified tailpiece region of human IgM
<400> 11
Pro Pro Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr
1 5 10 15
Cys Tyr
<210> 12
<211> 20
<212> PRT
<213> Mus musculus
<220>
<221> IgM tailpiece sequence
<400> 12
Gly Lys Phe Thr Leu Tyr Asn Val Ser Leu lie Met Ser Asp Thr Gly
1 5 10 15
Gly Thr Cys Tyr
20
<210> 13
<211> 18
<212> PRT
<213> Homo sapiens
<220>
<221> Tailpiece region of human IgA
<400> 13
Pro Thr His Val Asn Val Ser Val Val Met Ala Gin Val Asp Gly Thr
1 5 10 15
Cys Tyr
<210> 14
<211> 7
<212> PRT
<213> Homo sapiens
<220>
<221> Fc receptor CH2 domain
<400> 14
Glu Leu Leu Gly Gly Pro Ser

CLAIMS
1. A method for treatment of a mammalian subject for an autoimmune or
inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
comprising five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion
comprising two immunoglobulin G heavy chain constant regions;
wherein 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;
wherein the polymeric protein does not comprise a further immunomodulatory portion; or
an antigen portion that causes antigen-specific immunosuppression when administered
to the mammalian subject.
2. The method of Claim 1 wherein each polypeptide monomer unit comprises a
tailpiece region fused to each of the two immunoglobulin G heavy chain constant
regions; wherein the tailpiece region of each polypeptide monomer unit facilitates the
assembly of the monomer units into a polymer.
3. The method of Claim 2 wherein the tailpiece region is an Ig or IgA tailpiece, or
fragment or variant thereof.
4. The method of Claim 1 wherein each of the immunoglobulin G heavy chain
constant regions comprises an amino acid sequence having at least 90% sequence
identity to a native human immunoglobulin G 1 heavy chain constant region.
5. The method of Claim 1 wherein each of the immunoglobulin G heavy chain
constant regions comprises an amino acid sequence which comprises a cysteine residue
at position 309 according to the EU numbering system, and preferably also a leucine
residue at position 310.
6. The method of Claim 1 wherein each of the immunoglobulin G heavy chain
constant regions comprises an amino acid sequence which is modified compared to the
amino acid sequence of a native immunoglobulin G heavy chain constant region, to
modify the affinity of the Fc receptor binding portion for at least one Fc receptor.
7. The method of Claim 1 wherein each of the immunoglobulin G heavy chain
constant regions comprises an amino acid sequence which is modified compared to the
amino acid sequence of a native immunoglobulin G heavy chain constant region, to
increase the in vivo half life of the polymeric protein, suitably by increasing the affinity of
the Fc receptor binding portion for neonatal Fc receptor.
8. The method of Claim 1 wherein the autoimmune or inflammatory disease is
treatable with intravenous immunoglobulin (IVIG).
9. The method of Claim 1 wherein the autoimmune or inflammatory disease is an
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, and uveitis or Alzheimer's disease.
10. A method for treatment of a mammalian subject for an autoimmune or
inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
consisting of five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion
consisting of two immunoglobulin G heavy chain constant regions; and, optionally, a
polypeptide linker linking the two immunoglobulin G heavy chain constant regions as a
single chain Fc; and
wherein 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.
11. A method for treatment of a mammalian subject for an autoimmune or
inflammatory disease, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein
consisting of five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion and a
tailpiece region;
wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain
constant regions; and, optionally, a polypeptide linker linking the two immunoglobulin G
heavy chain constant regions as a single chain Fc;
wherein each modified human immunoglobulin G heavy chain constant region comprises
a cysteine residue which is linked via a disulfide bond to a cysteine residue of a modified
human immunoglobulin G heavy chain constant region of an adjacent polypeptide
monomer unit; and
wherein the tailpiece region is fused to each of the two modified human immunoglobulin
G heavy chain constant regions of the polypeptide monomer unit, and facilitates the
assembly of the monomer units into a polymer.
12. The method of Claim 11 wherein the tailpiece region is an Ig or IgA tailpiece, or
fragment or variant thereof.
13. A polymeric protein comprising five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion
comprising two immunoglobulin G heavy chain constant regions;
wherein 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;
wherein the polymeric protein does not comprise a further immunomodulatory portion; or
an antigen portion that causes antigen-specific immunosuppression when administered
to a mammalian subject;
wherein each polypeptide monomer unit does not comprise a tailpiece region fused to
each of the two immunoglobulin G heavy chain constant regions.
14. A polymeric protein consisting of five, six or seven polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion
consisting of two immunoglobulin G heavy chain constant regions; and, optionally, a
polypeptide linker linking the two immunoglobulin G heavy chain constant regions as a
single chain Fc; and
wherein 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.
15. A nucleic acid molecule comprising a coding portion encoding a polypeptide
monomer unit of a polymeric protein as defined in Claim 13 or Claim 14.