The present invention relates to proteins, and compositions comprising such proteins. The invention also relates to the medical uses of such proteins and compositions. In particular the proteins or compositiosn of the invention may be used in the prevention or treatment of autoimmune diseases or inflammatory diseases, or for the prevention or treatment of diseases mediated through binding of sialic acid dependent receptors, or as vaccines. The invention further relates to methods of preventing or treating autoimmune or inflammatory diseases, or diseasesmediated through binding of sialic acid dependent receptors, using such proteins. The invention also relates to nucleic acids encoding the proteins, as well as methods of manufacturing the proteins.

BACKGROUND

Autoimmune diseases (ADs) are common and affect 50 million American citizens alone. Intravenous immunoglobulin (IVIG) treatment involves the adminstration of purified immunoglobulin G, and is one of the most common treatments of ADs, with Food and Drug Administration (FDA) approval for a diverse range of diseases like idiopathic thrombocytopenia (ITP), Kawasaki disease, Guillain-Barre, dermatomyositis, and chronic inflammatory demyelinating polyneuropathy.

As 70% of the global supply (worth $5 billion in 2012) of IVIG is now used to treat ADs, it is increasingly becoming unavailable to patients that need it most, in particular individuals with primary immune deficiency where IVIG is used as replacement therapy.

The worldwide consumption of IVIG has increased over 300 fold since 1980 and currently 100 ton are consumed per annum. Supplies of IVIG within the NHS and globally are critically limited, meaning that patients with an urgent need for the drug are routinely deprived of it. There are also significant clinical limitations resulting from its dependence on human donors for manufacture, and from the fact that <5% of injected IVIG (correctly glycosylated and/or oligomeric-Fc) is therapeutically active leading to a requirement for high doses (2g/kg) when used in idiopathic thrombocytopenic purpura (ITP). Consequently, IVIG is expensive and adverse events due to excessive protein loading not uncommon.

Whereas some effector mechanisms of IgG relevant to autoimmune diseases may be F(ab')2-mediated, e.g. blocking/neutralization of receptors, cytokines, anaphylatoxins and pathogenic auto-antibodies via anti-idiotypic interactions, many anti-inflammatory functions are thought to be mediated by the Fc portion. They include FcRn saturation, blockade and modulation of FcyR expression, modulation of dendritic cell, B cells and T regulatory cell function and blockade/scavenging of complement components. IVIG suppresses harmful inflammation by engaging low-affinity inhibitory receptors and by forming immune-complexes (ICs) and/or dimers when injected in vivo that allow IVIG to interact with these receptors with greater strength (avidity), thus mediating more potent anti-inflammatory effects.

The problems noted above have led to a number of attempts to generate artificial agents, capable of expression on a large scale that can be used as replacements for human IgG in therapies, such as IVIG, for use in the treatment of autoimmune and inflammatory diseases.

Examples of such artificial agents that have been described to date include "SIFs" (selective immunomodulators of Fc-receptors), such as SIF-3, manufactured by Momenta, "stradomers" manufactured by Pfizer, and "hexa-Fc" an immunoglobulin-based hybrid protein produced by the current inventors. Each of the molecules produced in this manner has been designed to favour the formation of oligomeric structures that incorporate multiple Fc-receptor binding domains. This approach has been taken with a view to increasing avidity of binding of the artificial agents in the subjects to whom they are administered.

Cells carry various receptors that depend upon glycans comprising sialic acid for their binding. Examples of such sialic acid dependent receptors include SIGLEC-1 and SIGLEC-2. It is known that a range of diseases are mediated through binding to these sialic acid dependent receptors. For example, a number of infectious agents, such as retroviruses, bind to cells, and thus cause their associated infections, through binding to the cells' sialic acid dependent receptors.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a protein comprising two chimeric polypeptide chains; wherein each chimeric polypeptide chain comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions; and an immunoglobulin tailpiece region;

wherein the amino acid sequence and glycosylation of the tailpiece region is adapted, as compared to the sequence and glycosylation of wild-type immunoglobulin, to inhibit polymerisation of the protein.

Suitably, the protein of the invention comprises a cysteine at a residue which corresponds to residue 89 of SEQ ID NO:1 (which in turn, corresponds to residue 309 of human IgG).

In a second embodiment, the invention provides a composition comprising a protein according to the first aspect of the invention, wherein at least 95% of the protein of the first aspect of the invention incorporated in the composition is in monomeric form.

The term "monomeric" as used in the context of the present invention, is considered in more detail elsewhere in this disclosure.

In a third aspect, the invention provides a protein in accordance with the first aspect of the invention for use as a medicament. The protein for use in the third aspect of the invention may be provided in the form of a composition in accordance with the second aspect of the invention.

Proteins or compositions of the invention may be used as medicaments in the prevention and/or treatment of autoimmune or inflammatory diseases. Suitable examples of such diseases are considered elsewhere in the specification.

Alternatively, proteins or compositions of the invention may be used as medicaments in the prevention and/or treatment of diseases mediated through the binding of sialic acid dependent receptors. In an embodiment, the receptor may be selected from the group consisting of: SIGLEC-1 and SIGLEC-2. Suitable examples of such diseases include retroviral infections, as considered elsewhere in the specification.

In a fourth aspect, the invention provides a method of preventing or treating an autoimmune or inflammatory disease, the method comprising providing a therapeutically effective amount of protein in accordance with the first aspect of the invention to a subject in need of such prevention or treatment. The subject may be a human subject.

In a fifth aspect, the invention provides a method of preventing or treating a disease mediated through binding of sialic acid dependent receptors, the method comprising

providing a therapeutically effective amount of protein in accordance with the first aspect of the invention to a subject in need of such prevention or treatment. Suitably the subject is human. In an embodiment, the receptor may be selected from the group consisting of: SIGLEC-1 and SIGLEC-2. The disease may be an infection or an autoimmune disease. The disease may be a retroviral infection. The invention also provides corresponding medical uses.

The medical uses or methods of treatment of the third, fourth or fifth aspects of the invention may employ the proteins of the invention in intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG) therapy.

In a sixth aspect, the invention provides a nucleic acid encoding a protein in accordance with the first aspect of the invention.

In a seventh aspect, the invention provides a method of producing a protein in accordance with the first aspect of the invention, the method comprising expressing a nucleic acid in accordance with the sixth aspect of the invention in a host cell.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based upon the inventors' surprising finding that proteins of the invention, which remain monomeric in physiological conditions, are suitable for use in therapeutic applications such as IVIG and/or SCIG. This goes entirely against the expectations of those skilled in the art, since it had been widely considered desirable to produce oligomeric or polymeric Fc receptor-binding molecules, with a view to increasing the effectiveness of artificial agents generated for use in IVIG and/or SCIG.

The ability of a molecule to bind to glycan receptors, in particular Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), is associated with therapeutic utility in IVIG or SCIG. Previously it has been found that less than 5% of native, monomeric IgG molecules are correctly glycosylated in a way that allows them to interact with DC-SIGN, thus rendering these molecules unsuitable for therapeutic purposes.

Oligomeric or polymeric Fc receptor-binding molecules have increased avidity for DC-SIGN, which is advantageous in terms of their ability to bind to this receptor. However, oligomeric or polymeric Fc receptor-binding molecules previously described in the prior art have been shown to activate the complement cascade. This is a significant disadvantage in a potential therapeutic molecule, due to the risk of adverse consequences, such as anaphylactic shock.

The inventors have found that an adaptation of the amino sequence and glycosylation of the tailpiece region of the proteins of the invention, results in a monomeric protein glycosylated in a way which allows the protein of the invention to bind to DC-SIGN and sialic acid dependent receptors such as SIGLEC-1 , and thus exert therapeutic utility in IVIG or SCIG. Furthermore, the proteins of the invention, surprisingly, do not lead to the activation of the complement cascade.

Proteins of the invention feature adaptation of the amino acid sequence and glycosylation of the tailpiece region, as compared to the sequence and glycosylation found in wild-type immunoglobulins from which they are derived, and these inhibit polymerisation of the hybrid proteins of the invention.

Suitably the adaptation of the amino acid sequence is loss of a cysteine residue. Suitably the adaptation may be loss of a single cysteine residue as compared to the wild-type sequence. Alternatively, the loss may be of multiple cysteine residues as compared to the wild-type sequence.

The loss may comprise loss of the cysteine corresponding to residue 248 of SEQ ID NO:1. In a suitable embodiment, a protein of the invention may also have loss of the cysteine residue corresponding to residue 89 of SEQ ID NO: 1.

One or more cysteine residues may be lost, as compared to the sequence of the wild-type immunoglobulin tailpiece (such as the IgM tailpiece), or protein of SEQ ID NO: 1 , by their substitution or deletion. In a suitable embodiment of a substitution, a cysteine residue, such as the cysteine residue corresponding to residue 248 of SEQ ID NO: 1 , is replaced with a different amino acid. The cysteine residue by may replaced with any amino acid residue (for example an alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine residue). Suitably, cysteine residues, such as the cysteine residue corresponding to residue 248 of SEQ ID NO: 1 , is replaced with an alanine residue.

As referred to above, proteins of the invention also incorporate an adaptation of glycosylation of the tailpiece region that inhibits their polymerisation. The inventors have found that the glycans attached to glycosylation sites of proteins of the invention (as exemplified by SEQ ID NO:2) are larger than those on relevant control proteins (such as the protein of SEQ ID NO: 1).

In a suitable embodiment, proteins of the invention may also comprise an adaptation of glycosylation of the immunoglobulin G derived sequence (such as the immunoglobulin G heavy chain constant region Cy2) that inhibits their polymerisation. Such a glycosylation may be found, for example, at a residue corresponding to N77 of SEQ ID NO: 2. Proteins of the invention incorporating such an adaptation may be of particular utility in application requiring and making use of the ability to bind sialic acid-dependent receptors.

Suitably, the immunoglobulin G derived sequence of the proteins of the invention may comprise an artificial glycosylation site. Such an artificial glycosylation site may involve a substitution at a residue corresponding to residue D1 of SEQ ID NO: 1 (in turn corresponding to D221 of human IgG). Suitably, such an artificial glycosylation may be obtained by an aspartic acid to asparagine substitution (for example a D1 N substitution in SEQ ID NO: 1 , corresponding to a D221 N substitution of human IgG). An example of a protein of the invention comprising such a modification is shown in SEQ ID NO: 17, which corresponds to SEQ ID NO: 2, save for substitution of residue D1 with N. The protein of SEQ ID NO: 17 is a particularly useful example of a protein of the invention. It represents an embodiment that is highly suited to medical uses and methods in which it is desired for proteins of the invention to bind to sialic acid dependent receptors (for example to prevent or treat diseases mediated through binding of sialic acid dependent receptors).

It will be appreciated that artificial glycosylation constitutes further adaptation of the amino acid sequence and glycosylation of proteins of the invention to inhibit their polymerization.

Without wishing to be bound by any hypothesis, the inventors believe that the combination of loss of cysteine residues that would otherwise be able to form disulphide bridges between protein monomers, in combination with the capacity for larger glycans to be added at the glycosylation sites present within the monomers, significantly inhibits polymerisation of the proteins of the invention. As discussed elsewhere in the invention, these changes are sufficient to decrease the proportion of the proteins occurring in polymeric form from greater than 80% to less than <1 %. The remarkable extent of this reduction could not be predicted prior to the results disclosed for the first time in the present specification.

Since polymerisation of the proteins of the invention is inhibited through a combined impact of adaptations of the amino acid sequence and glycosylation of the tailpiece, each of these individual modifications can be relatively minimal, while still achieving a marked inhibition in overall levels of polymerisation. The ability to utilise minimal departures from the wild-type sequences in this manner decreases the likelihood of the proteins of the invention inducing adverse immunogenic responses in subjects to whom they are administered, and this provides a further notable advantage of the proteins of the invention.

A further advantage of the modification of glycosylation observed is that, the larger and more complex glycans present are more likely to terminate in sialic acid (neuraminic acid). Glycans terminating in this manner are known to interact with DC-SIGN, and enhanced binding to DC-SIGN and SIGLEC-1 is observed in respect of proteins of the invention.

Additionally, the inventors have found that the introduction of an artificial glycosylation site is able to give rise to a protein with greater sialylation than a protein without such an artificial glycosylation site, and thus may yield a protein with greater efficacy for use in sialic acid dependent therapies.

In light of the above, it will also be appreciated that the presence of an artificial glycosylation site, in particular at residue 1 of SEQ ID NO: 2 (such as in the protein of SEQ ID NO:17), may enable binding of the proteins of the invention to sialic acid dependent receptors including SIGLEC-1 , (also known as sialoadhesisn), as well as DC-SIGN.

Accordingly, such proteins may have a therapeutic effect in a number of diseases where binding to sialic acid dependent receptors, such as SIGLEC-1 may be desirable. For example, proteins of the invention may be used as medicaments in diseases in which it is desirable to compete with, and thereby inhibit or prevent, the binding of other molecules to sialic acid dependent receptors, such as SIGLEC-1. Merely by way of example, binding of proteins of the invention to sialic acid dependent receptors such as SIGLEC-1 may have a therapeutic effect in inhibiting retrovirus binding to these receptors, thus preventing or treating retrovirus infections (such as HIV or T-cell leukaemia virus infections).

The presence of an additional glycan at the artificial glycosylation site may also confer a further advantage as it may increase the protein's stability. Currently, in order to increase immunoglobulin stability, immunoglobulins are often chemically glycosylated, for example by in vitro enzymatic or non-enzymatic reactions. However, the presence of an artificial glycosylation site allows such modifications to be introduced by cells expressing the proteins, and thus may eliminate the need for this additional step of chemical glycosylation. As a result, the proteins of the invention may be produced in a more cost and time effective manner than traditional agents used in IVIG treatment.

Various aspects and embodiments of the invention will now be further described in the following paragraphs.

Exemplary proteins of the invention

An example of a protein of the invention is set out in SEQ ID NO: 2. A further exemplary protein of the invention is set out in SEQ ID NO: 17. A protein of the invention may comprise SEQ ID NO: 2 or SEQ ID NO: 17. In a suitable embodiment, a protein of the invention may consist of SEQ ID NO: 2 or SEQ ID NO: 17.

The chimeric polypeptide of SEQ ID NO: 2 comprises residues 221 to 447 of human lgG1 (corresponding to residues 1 to 227 of SEQ ID NO: 2) in combination with residues based upon, and adapted from, 558 to 576 of the tailpiece of human IgM (corresponding to residues 232 to 249 of SEQ ID NO: 2). The wild type immunoglobulin tailpiece sequence is adapted in SEQ ID NO: 2 to inhibit polymerisation of the protein of the invention.

SEQ ID NO: 17 corresponds directly to SEQ ID NO: 2, save for the presence of a D to N substitution at residue 1 of SEQ ID NO: 17. As discussed above, this substitution introduces a new glycosylation site in the protein of SEQ ID NO: 17.

It will be appreciated that in chimeric polypeptides, where the full IgG or IgM sequences are not present, numbering of residues based upon the full-length IgG or IgM molecules is no longer informative. Accordingly, we will also refer in this disclosure to a reference chimeric protein sequence, which is set out as SEQ ID NO: 1. This sequence represents a single chimeric polypeptide chain. When referring to this sequence, Cys575 of the full length IgM sequence is renumbered as Cys248 of the fusion protein (the 248th residue of SEQ ID NO: 1).

For the avoidance of doubt, a protein consisting of chimeric protein chains having the sequence set out in SEQ ID NO: 1 will not constitute a protein of the invention, since it will not incorporate the requisite adaptations to inhibit polymerisation.

The inventors' have surprisingly found, that SEQ ID NO: 2 may encode monomeric proteins of two different sizes, specifically -53 kDa and -58 kDa. Without wishing to be bound to any hypothesis, the inventors' believe that differentially glycosylated at residues N77 of SEQ ID NO: 2 (corresponding to N297 of human lgG1) in the immunoglobulin G heavy chain constant region (such as Cy2 domain), and at N236 of SEQ ID NO: 2 (corresponding to N563 of human IgM) in the immunoglobulin tailpiece give rise to the two sizes of monomeric proteins.

In addition to the Fc receptor binding portion and the tailpiece region, the proteins of the invention may further comprise a hinge region and/or a spacer region.

In a suitable embodiment, the proteins of the present invention may be conjugated to a therapeutic payload. Suitable therapeutic payloads are described elsewhere in this specification. Suitably the payload may be conjugated to the Fc receptor binding portion of the protein of the invention. Alternatively, it may be conjugated to the hinge region of the protein of the invention.

In a suitable embodiment, a hinge region may be located at the N-terminus of the Fc receptor binding portion. The hinge region may be a natural or synthetic hinge region.

In a suitable embodiment, the hinge region is natural. A natural hinge region is one that is naturally found between the Fc and Fab portion of an antibody. A natural hinge region may be derived from the same species as the Fc receptor binding portion. Alternatively, it may be derived from a different species.

In a suitable embodiment, a natural hinge region may be derived from an antibody of the same class or subclass as the Fc receptor binding portion. Alternatively, it may be derived from an antibody of a different class or subclass as the Fc receptor binding portion.

In a suitable embodiment, the hinge region is derived from IgGl More suitably, the hinge region may be derived from human lgG1. By way of example, in the protein of the invention according to SEQ ID NO:2, comprises a hinge region derived from human.

In a suitable embodiment, the N-terminus of the hinge region may be glycosylated in a way so as to inhibit polymerisation of the protein of the present invention. Suitably, the glycosylation may be at a position corresponding to residue 1 of SEQ ID NO: 1 or SEQ ID NO: 2 (as exemplified by the protein of SEQ ID NO: 17). Glycosylation of the hinge region may be beneficial as it may result in an exposed glycan, which may modify the function of the protein of the invention. By way of example, and as further explained in the Examples section of this description, glycosylation of the hinge region may reduce the protein's interactions with Fc-gamma receptors, while increasing interactions with sialic acid dependent receptors such as SIGLEC-1.

In another embodiment, the hinge region is synthetic. A synthetic hinge region is one that differs in length or sequence from a hinge region which is found naturally. By way of example, the difference in length between a synthetic and natural hinge region may be as a result of the addition or deletion of residues in the synthetic hinge region (for example addition or deletion of cysteine residues). A difference in sequence between a synthetic and natural hinge region may be as a result of a substitution of one or more residues in the synthetic hinge region (for example substitution of a cysteine residue with another residue such as serine or alanine).

In a suitable embodiment, a hinge region may be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty five, at least thirty, or more amino acid residues long.

By way of example and not limitation, a protein of the invention may comprise a hinge region, wherein the hinge region has a sequence selected from the group consisting of: VPSTPPTPSPSTPPTPSPS (SEQ ID NO: 8), VPPPPP (SEQ ID NO: 9), EPKSCDKTHTCPPCP (SEQ ID NO: 10), ERKCCVECPPCP (SEQ ID NO: 1 1), ESKYGPPCPSCP (SEQ ID NO: 12), CPPC (SEQ ID NO: 13), CPSC (SEQ ID NO: 14), and SPPC (SEQ ID NO: 15). Other suitable natural and synthetic hinges will be known to those skilled in the art.

The presence of a hinge region may be especially desirable in embodiments where the protein of the invention is conjugated to a therapeutic payload. It will be appreciated that such a hinge region may increase the distance between the Fc receptor binding portion and the therapeutic payload, if present. When the therapeutic payload is conjugated to the protein of the invention, increased distance between the Fc receptor binding portion and the therapeutic payload may be desirable in order to provide sufficient space for the attachment of a glycan molecule to a glycosylation site. Suitably, the hinge region provides space for the attachment of a glycan molecule to an artificial glycosylation site (for example at residue 1 of SEQ ID NO: 1 , as found in SEQ ID NO:17).

It will be also be appreciated, that the presence of a hinge region may be desirable for the insertion and/or attachment of an additional N-linked glycosylation site in the protein of the invention.

As touched upon above, the protein of the invention may comprise a spacer region. Suitably, the spacer region may be between the Fc receptor binding portion and the tailpiece region.

A suitable spacer region may be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acid residues long. More suitably, the spacer region may be four amino acid residues long. In the exemplary protein of the invention as set out by SEQ ID NO: 2, the spacer region may be found at residues 228 to 231.

Suitably the spacer region may have the sequence LVLG (SEQ ID NO: 16).

The presence of a spacer region may improve the disposition of the tailpiece carbohydrate (for example corresponding to amino acid residue N236 of the protein if the invention as set out in SEQ ID NO: 1 or SEQ ID NO:2) and thus improve interactions with glycan receptors without compromising interactions mediated through the Fc constant domains.

Adaptation of tailpiece amino acid sequence to inhibit polymerisation

Immunoglobulin tailpieces for incorporation in the proteins of the invention may be based upon any immunoglobulin molecule. Suitably a tailpiece may be based upon the tailpiece of an immunoglobulin selected from the group consisting of: IgM, IgA, and IgE.

A tailpiece based upon that of IgM is particularly suitable for incorporation in the proteins of the invention. Exemplary adaptations are described herein with reference to the IgM tailpiece (which is incorporated in the reference protein of SEQ ID NO: 1 , and the exemplary proteins of the invention of SEQ ID NO: 2 or SEQ ID NO:17). It will be appreciated that tailpieces of other immunoglobulins may be adapted at residues corresponding to those exemplified in respect of IgM. Furthermore, tailpieces derived from immunoglobulins other than IgM may be adapted in the same manner as described in respect of IgM.

Tailpieces suitable for incorporation in the proteins of the invention may, as long as they comprise relevant adaptations, share at least 55% identity with a native immunoglobulin tailpiece, such as the IgM tailpiece. Indeed a suitable tailpiece, as long as suitably adapted, may share at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more identity with the sequence of a corresponding portion of a native immunoglobulin tailpiece.

In particular, tailpieces suitable for incorporation in the proteins of the invention may, as long as they comprise the adaptations found in SEQ ID NO:2, share at least 55% identity with the IgM-derived sequences of SEQ ID NO: 2 (i.e. amino acid residues 232-249 of SEQ ID NO: 2). Suitably a tailpiece for incorporation in a protein of the invention may share at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more identity with residues 232-249 of SEQ ID NO:2.

For the avoidance of doubt, a protein that comprises the wild-type amino acid sequence of an immunoglobulin tailpiece, such as the IgM tailpiece, without any alteration, whether by substitution or deletion, will not constitute a protein of the invention.

Adaptation of tailpiece glycosylation to inhibit polymerisation

Proteins of the invention must include at least one glycosylation site, and may include two or more glycosylation sites, within the tailpiece. These may be naturally occurring glycosylation sites retained from the native immunoglobulin tailpiece sequence. Alternatively, the proteins of the invention may include artificially introduced glycosylation sites in the tailpiece region, or combinations of naturally occurring and artificial sites. The inventors have found that when glycosylation sites are absent (such as in the control protein of SEQ ID NO:4) inhibition of polymerisation is much reduced, and so polymer formation increases.

For the purposes of the present disclosure, a protein in which the glycosylation of the immunoglobulin tailpiece, such as the IgM tailpiece, is not altered as compared to the glycosylation observed in respect of the wild-type tailpiece, will not constitute a protein of the invention.

Adaptation of hinge region amino acid sequence to inhibit polymerisation

Hinge regions for incorporation in the proteins of the invention may be based upon any immunoglobulin molecule. Suitably a hinge region be based upon the hinge region of an immunoglobulin selected from the group consisting of: IgG, IgA, IgE, IgD and IgM. More suitably, the hinge region of an IgG immunoglobulin may be selected from the group consisting of lgG1 , lgG2, lgG3 and lgG4.

A hinge region based upon that of immunoglobulin lgG1 is particularly suitable for incorporation in the proteins of the invention. Exemplary adaptations of a hinge region are described herein with reference to the lgG1 hinge region (which is incorporated in the reference protein of SEQ ID NO: 1 , and the exemplary proteins of the invention of SEQ ID NO: 2 and SEQ ID NO: 17).

It will be appreciated that hinge regions of other IgG immunoglobulins may be adapted at residues corresponding to those exemplified in respect of lgG1. Furthermore, hinge regions derived from immunoglobulins other than IgG may be adapted in the same manner as described in respect of lgG1.

Hinge regions suitable for incorporation in the proteins of the invention may, as long as they comprise relevant adaptations, share at least 55% identity with a native immunoglobulin hinge region, such as the IgG hinge region. Indeed a suitable hinge region, as long as suitably adapted, may share at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more identity with the sequence of a corresponding portion of a native hinge region.

In particular, hinge regions suitable for incorporation in the proteins of the invention may, as long as they comprise the adaptations found in SEQ ID NO:2, share at least 55% identity with the IgG hinge region derived sequences of SEQ ID NO: 2. Suitably a hinge region for incorporation in a protein of the invention may share at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more identity with the residues of the hinge region of SEQ ID NO:2.

Adaptation of hinge region glycosylation to inhibit polymerisation

Proteins of the invention may include a glycosylation site within the hinge region. The glycosylation site may be a naturally occurring glycosylation site retained from a native immunoglobulin hinge region sequence. Alternatively, the proteins of the invention may include an artificially introduced glycosylation site in the hinge region, or combinations of naturally occurring and artificial sites. The inventors have found that when glycosylation sites are absent (such as in the control protein of SEQ ID NO: 4) inhibition of polymerisation is much reduced, and so polymer formation may increase.

Inhibition of polymerisation of proteins of the invention

In proteins of the invention, the amino acid sequence and glycosylation of the tailpiece region, and optionally, the hinge region is adapted, when compared to the sequence and glycosylation of the corresponding wild-type tailpiece (such as the IgM tailpiece), and native hinge region respectively, to inhibit polymerisation of the protein. Inhibition of polymerisation of such proteins may be demonstrated by either a decrease in the proportion of protein present in a polymeric form, or an increase in the proportion of the protein that is present in a monomeric form. This may be assessed with reference to the proportion of polymeric or monomeric protein found in an appropriate control protein. Such an appropriate control protein may comprise a wild-type tailpiece, for example the IgM tailpiece, and optionally, may comprise a native hinge region.

In a control protein (SEQ ID NO:1) lacking the adaptations of the proteins of the invention (as exemplified by SEQ ID NO:2) monomers make up less than 20% of the total protein. In contrast, the inventors have found that more than 90% of a protein of the invention (such as SEQ ID NO:2) is present in monomeric form under physiological conditions.

Thus in the case of a protein, such as a protein of the invention as exemplified by SEQ ID NO:2 or SEQ ID NO:17, in which the amino acid sequence and glycosylation of the tailpiece region and hinge region are adapted as compared to the wild type sequence, the adaptation may be demonstrated to be one that inhibits polymerisation if 90% or more of the protein is present in monomeric form under physiological conditions. Indeed, in a suitable embodiment, inhibition of polymerisation may result in 95% or more of a protein being present in monomeric form, for example, 96% or more, 97% or more, 98% or more, or even 99% or more. In a suitable embodiment, inhibition of polymerisation may result in substantially all of a protein of the invention being present in monomeric form under physiological conditions.

Suitable methods by which the proportion of polymeric or monomeric protein in a sample may be determined are described in the Examples section later in this specification. Briefly, these include size-exclusion chromatography and SDS-PAGE acrylamide gradient gels.

IgG sequences suitable for use in the proteins of the invention

The proteins of the invention incorporate two immunoglobulin G heavy chain constant regions. In a suitable embodiment, the immunoglobulin G heavy chain constant regions employed in the monomeric proteins of the invention are derived from an immunoglobulin selected from the group consisting of: lgG1 ; lgG2; lgG3; and lgG4. In particular, the immunoglobulin G heavy chain constant regions may be derived from lgG1.

It will be appreciated that as long as they meet the requirement of forming an Fc receptor binding portion, the immunoglobulin G heavy chain constant regions utilised in proteins of the invention may include an alteration in their sequence as compared to the native sequences from which they are derived. Merely by way of example, a suitable protein of the invention may utilise IgG derived sequences that share at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the relevant native IgG sequence from which they are derived.

Monomers of proteins of the invention

For the avoidance of doubt, in the context of the present disclosure, references to a "monomer" of a protein of the invention are intended to cover a molecule made up of two chimeric polypeptide chains associated with one another. Suitably, the two chimeric polypeptide chains may be linked by an inter-disulphide bond formed between residue Cys226 and Cys229 of SEQ ID NO: 1.

Thus it can be seen that, for present purposes, a "trimer" would be made up of three "monomers" as referred to above - a total of six chimeric polypeptide chains. A "hexamer" would consist of six monomers, and hence a total of twelve chimeric polypeptide chains.

Compositions and pharmaceutical compositions of the invention

The second aspect of the invention provides a composition comprising a protein according to the first aspect of the invention. In such a composition, at least 95% of the protein of the first aspect of the invention incorporated in the composition is in monomeric form.

Suitably a composition of the second aspect of the invention may be a pharmaceutical composition, in which the protein is provided with a pharmaceutically acceptable carrier.

In a suitable embodiment of a composition of the invention, whether a pharmaceutical composition or otherwise, at least 96% or at least 97% of the protein of the first aspect of the invention incorporated in the composition is in monomeric form. Indeed in a suitable embodiment, at least 98% or at least 99% of the protein of the first aspect of the invention incorporated in the composition is in monomeric form. Suitably substantially all of the protein of the first aspect of the invention incorporated in such a composition may be in monomeric form.

Medical uses of the proteins of the invention

The proteins of the invention, for example provided in a composition of the invention, may be used as a medicament.

Proteins of the invention may, for example, be used as medicaments in IVIG or SCIG. Such medical uses of the proteins and compositions are of particular utility in the prevention or treatment of autoimmune or inflammatory diseases. Medical use of the proteins of the invention in this manner may be effective, irrespective of whether or not they are conjugated to a therapeutic payload.

Proteins of the invention may be used as medicaments for the prevention or treatment of diseases mediated through binding of sialic acid-dependent receptors.

As mentioned elsewhere in this specification, proteins of the invention, in particular proteins comprising the artificial glycosylation site at residue 1 of SEQ ID NO:2 or SEQ ID NO: 17, have the ability to bind sialic acid-dependent receptors, such as SIGLEC-1 , and thereby prevent other molecules from binding to the receptor. The inventors believe that the ability of the proteins to bind SIGLEC-1 and other sialic acid-dependent receptors is a result of their greater sialylation.

Accordingly, the proteins of the invention may be used as medicaments in diseases in which preventing the binding of other molecules to sialic acid dependent receptors may have a therapeutic effect. Merely by way of example, preventing binding to SIGLEC-1 may have a therapeutic effect in retrovirus infections (such as HIV or T-cell leukaemia virus infections), or other conditions in which infectious agents bind via SIGLEC-1. Accordingly, proteins of the invention, and in particular proteins comprising artificial glycosylation site corresponding to that found at residue 1 of SEQ I D NO:2 or SEQ I D NO: 17, may be used in the prevention or treatment of infections. Suitably proteins of the invention may be used in the prevention or treatment of retrovirus infections.

Proteins of the invention comprising or consisting of SEQ ID NO:17 are particularly suited for the medical uses described above.

Other suitable examples of such diseases, which may benefit from prevention or treatment through medical use of the proteins of the invention, are considered below.

As mentioned elsewhere in this specification, the protein of the invention may be conjugated to a therapeutic payload. The term "therapeutic payload" as used herein refers to a compound which itself has a therapeutic effect. The therapeutic effect of a therapeutic payload may be in addition to, or independent of, the therapeutic effect of the protein of the invention.

Further medical uses of the proteins of the invention may be selected with reference to a therapeutic payload conjugated to such proteins. A suitable therapeutic payload may be selected from the group consisting of an immune modulator, a drug, a protein, a carbohydrate, and a nucleic acid.

A suitable immune modulator may upregulate or downregulate components of the immune system.

A protein of the invention conjugated to an immune modulator which upregulates components of the immune system may be useful as a vaccine. By way of example an immune modulator which may be useful as a vaccine may be a pathogen-associated molecular pattern (PAMP) molecule or an antigen. Accordingly, the present invention provides the use of proteins of the invention as vaccines.

A protein of the invention conjugated to an immune modulator which down regulates the components of the immune system may be useful as a medicament for autoimmune diseases, for example rheumatoid arthritis.

An examples of such an immune modulator which down regulates the components of the immune system is erythropoietin. Accordingly, it will be appreciated that in a suitable embodiment erythropoietin may be conjugated to a protein of the invention. Such a conjugated protein may be used in the prevention or treatment of an autoimmune disease.

The term "drug" as used herein refers to a compound with therapeutic activity, for example a small molecule, which may be conjugated to a protein of the invention. Merely by way of example, a suitable drug therapeutic payload may be one, such as monomethyl auristatin E, which may be useful in the treatment of cancer. Suitably, the drug, such as monomethyl auristatin E, may be further conjugated to an antibody. Accordingly, a protein of the invention may be conjugated to an anti-cancer drug, such as monomethyl auristatin E. Such a conjugated protein may be used in the prevention or treatment of cancer.

Merely by way of example, a suitable protein therapeutic payload for conjugation to a protein of the invention may be a cytokine receptor. Cytokine receptors may be useful for inhibiting disease causing cytokines, by for example, binding such disease causing cytokines, and thereby preventing them from pathogenically binding to cells.

A suitable carbohydrate payload to be conjugated to a protein of the invention may be, for example, hyaluronic acid.

A suitable nucleic acid payload to be conjugated to a protein of the invention may be, for example, unmethylated CpG oligodeoxynucleotide. Proteins of the invention conjugated in this manner are suitable for medical use as immunostimulants.

Methods of treatment using the proteins of the invention

The proteins of the invention, for example provided in a composition of the invention, may be used as a medicament. The proteins may be conjugated to a therapeutic payload. Alternatively, they may be not conjugated to a therapeutic payload.

Such medical uses of the proteins and compositions thereof are of particular utility in the prevention or treatment of autoimmune or inflammatory diseases, whether or not the proteins are conjugated to a therapeutic payload.

Additionally, as already mentioned, the inventors have surprisingly found that the proteins of the invention, in particular proteins comprising the artificial glycosylation site at residue 1 of SEQ ID NO:2 (such as the protein of SEQ ID NO: 17), have the ability to bind sialic acid dependent receptors, for example, SIGLEC-1 receptors. The proteins may thereby prevent other molecules from binding to the receptor, or may be used to trigger such receptors for therapeutic effect.

Therefore, proteins of the invention which are not conjugated to therapeutic payloads may be particularly useful in the prevention or treatment of diseases in which preventing the binding of other molecules to SIGLEC-1 may have a therapeutic effect. Merely by way of example, preventing binding to sialic acid-dependent receptors such as SIGLEC-1 may have a therapeutic effect in prevention or treatment of infections, such as retrovirus infections (such as Human Immunodeficiency Virus or T-cell Leukaemia Virus infections).

Other suitable examples of such diseases, which may benefit from prevention or treatment through medical use of the proteins of the invention, are considered below.

The subject may be provided with a protein of the invention by any technique through which the subject will ultimately receive a therapeutically effective amount of the protein of the invention.

Thus, in a suitable embodiment the subject may be provided directly with the protein of the invention. In such an embodiment the subject may, for example, be provided with a composition of the invention comprising the protein of the invention in monomeric form.

In another embodiment the subject may be provided indirectly with the monomeric protein. By way of example, in such an embodiment, a nucleic acid according to the sixth aspect of the invention (a nucleic acid encoding a protein of the invention) may be administered to the subject, and the therapeutically effective amount of the protein of the invention provided by expression of the nucleic acid to yield the protein. Accordingly, in a seventh aspect, the present invention provides a nucleic acid in accordance with the sixth aspect of the invention for use as a medicament. The medical use of nucleic acids of the invention in this manner may be of benefit in the applications described with reference to the medical uses of proteins of the invention

Nucleic acids of the invention

The sixth aspect of the invention provides nucleic acids that encode the proteins of the invention. In a suitable embodiment, the nucleic acids may encode a chimeric polypeptide, wherein the cysteine corresponding to that at position 575 of IgM (equivalent to C248 of SEQ ID NO: 1) is lost. In such an embodiment the cysteine residue may be substituted by an alanine residue.

The nucleic acid of the invention may be a DNA molecule encoding a protein of the invention. Alternatively, the nucleic acid of the invention may be an RNA molecule, encoding a protein of the invention.

Suitably, a nucleic acid of the invention may comprise SEQ ID NO:3, which encodes a polypeptide of SEQ ID NO: 2.

In a suitable embodiment the nucleic acid of the invention may share at least 70% identity with SEQ ID NO: 3, at least 75% identity with SEQ ID NO: 3, at least 80% identity with SEQ ID NO: 3, at least 85% identity with SEQ ID NO: 3, at least 90% identity with SEQ ID NO: 3, at least at least 95% identity with SEQ ID NO: 3, at least 96% identity with SEQ ID NO: 3, at least 97% identity with SEQ ID NO: 3, at least 98% identity with SEQ ID NO: 3, or at least 99% identity with SEQ ID NO: 3.

It will be appreciated the nucleic acids of the invention may be incorporated in larger nucleic acid sequences, which will comprise regions that do not encode the monomeric proteins of the invention. Merely by way of example, a nucleic acid of the invention may be incorporated in an expression plasmid, such as pFUSE-hlgG1-Fc-TP-LH309/310CL or pFUSE-hlgG1-FC-TP-L310H.

Production of proteins of the invention

The seventh aspect of the invention provides a method of producing a protein in accordance with the first aspect of the invention. These methods comprise expressing a nucleic acid in accordance with the sixth aspect of the invention in a host cell.

WE CLAIMS

1. A protein comprising two chimeric polypeptide chains; wherein each chimeric polypeptide chain comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions; and an immunoglobulin tailpiece region;

wherein the amino acid sequence and glycosylation of the tailpiece region is adapted, as compared to the sequence and glycosylation of wild-type immunoglobulin, to inhibit polymerisation of the protein.

2. A protein according to claim 1 , wherein the adaptation of the amino acid sequence is loss of a cysteine residue.

3. A protein according to claim 2, wherein the loss of a cysteine residue comprises loss of the cysteine corresponding to residue 248 of SEQ ID NO:1.

4. A protein according to claim 3, wherein the cysteine residue corresponding to residue 248 of SEQ ID NO: 1 , is replaced with an alanine residue.

5. A protein according to any of claims 2 to 4, wherein the loss of a cysteine residue comprises loss of the cysteine corresponding to residue 89 of SEQ ID NO: 1.

6. A protein according to any preceding claim, wherein the glycans attached to glycosylation sites of the proteins are larger than those found on relevant control proteins.

7. A protein according to any preceding claim, wherein the proportion of glycans that terminate in sialic acid (neuraminic acid) is larger than the proportion of such glycans found on relevant control proteins.

8. A protein according to any preceding claim, wherein the tailpiece is based upon the tailpiece of an immunoglobulin selected from the group consisting of: IgM, IgA, and IgE.

9. A protein according to claim 8, wherein the tailpiece is based upon the tailpiece of IgM.

10. A protein according to any preceding claim, wherein the tailpiece shares at least 70% identity with a native immunoglobulin tailpiece.

1 1. A protein according to any preceding claim, wherein the tailpiece shares at least 70% identity with amino acid residues 232-249 of SEQ ID NO:2.

12. A protein according to claim 1 1 , wherein the tailpiece shares at least 90% identity with amino acid residues 232-249 of SEQ ID NO:2.

13. A protein according to any preceding claim, wherein the immunoglobulin G heavy chain constant regions are derived from an immunoglobulin selected from the group consisting of: lgG1 ; lgG2; lgG3; and lgG4.

14. A protein according to claim 13, wherein the immunoglobulin G heavy chain constant regions are derived from lgG2.

15. A protein according to any preceding claim, wherein the IgG derived sequences that share at least 70% sequence identity with the native IgG sequence from which they are derived.

16. A protein according to any preceding claim comprising SEQ ID NO: 2.

17. A protein according to any preceding claim consisting of SEQ ID NO: 2.

18. A protein according to any of claims 1 to 15 comprising SEQ ID NO: 17.

19. A protein according to claim 18 consisting of SEQ ID NO: 17.

20. A protein in accordance with any of claims 1 to 19 for use as a medicament.

21. A protein for use in accordance with claim 20, for use in intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG) therapy.

22. A protein for use in accordance with claim 20 or claim 21 , for use in the prevention and/or treatment of autoimmune or inflammatory diseases.

23. A protein for use in accordance with any of claims 20 to 22, for use in the prevention and/or treatment of autoimmune or inflammatory diseases selected from the group consisting of: autoimmune cytopenias, Guillain-Barre syndrome, myasthenia gravis, anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis.

24. A protein for use according to claim 20 for use as a vaccine.

25. A protein for use according to claim 24, wherein the protein is conjugated to an immune modulator.

26. A protein in accordance with either claim 18 or claim 19 for use in the prevention or treatment of a disease mediated through binding of sialic acid-dependent receptors.

27. A protein according to claim 26 for use in the prevention or treatment of infection.

28. A protein according to claim 27 for use in the prevention or treatment of a retroviral infection.

29. A composition comprising a protein according to any of claims 1 to 28, wherein at least 95% of the protein incorporated in the composition is in monomeric form.

30. A method of preventing or treating an autoimmune or inflammatory disease, the method comprising providing a therapeutically effective amount of protein in accordance with any of claims 1 to 19 to a subject in need of such prevention or treatment.

31. A method according to claim 30, wherein the subject is a human subject.

32. A method according to claim 30 or claim 31 , wherein the proteins is provided in intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG) therapy.

33. A method of preventing or treating a disease, the method comprising providing a therapeutically effective amound of a protein in accordance with any of claims 1 to 19 as a vaccine to a subject in need of such treatment.

34. A method according to claim 33, wherein the protein is conjugated to an immune modulator.

35. A method of preventing or treating a disease mediated through binding of sialic acid-dependent receptors, the method comprising providing a therapeutically effective amount of a protein in accordance with any of claims 1 to 19 as a vaccine to a subject in need of such treatment.

36. A method according to claim 35, wherein the disease is an infection.

37. A method according to claim 36, wherein the infection is a retroviral infection.

38. A method according to any of claims 35 to 37, wherein the protein is as defined in claim 18 or claim 19.

39. A nucleic acid encoding a protein according to any of claims 1 to 19.

40. A nucleic acid according to claim 39 for use as a medicament.

41. A method of producing a protein according to any of claims 1 to 17, the method comprising expressing a nucleic acid in accordance with claim 39 in a host cell.