GB2572008A - Murine antibodies - Google Patents

Abstract

 A mouse fusion protein comprising two chimeric polypeptide chains each comprising two murine IgG heavy chain chains having an immunoglobulin tailpiece from, or derived from, a non-native immunoglobulin and wherein the amino acid sequence of each of the IgG heavy chain constant regions comprises an amino acid modification (e.g. M84C) which promotes the multimerisation or polymerisation of the fusion protein. Preferably the tailpiece is human and is derived from IgM or IgA and has a substitution or deletion at residue C17. Preferably, each chimeric chain comprises one or more murine hinge regions and a targeting moiety such as an ntigen binding region, small molecule or nucleic acid. The invention further relates to the uses of the fusion protein to detect a target molecule in a sample, preferably as a means for diagnosis such as blood group haemagglutination.

Description

(57) A mouse fusion protein comprising two chimeric polypeptide chains each comprising two murine IgG heavy chain chains having an immunoglobulin tailpiece from, or derived from, a non-native immunoglobulin and wherein the amino acid sequence of each of the IgG heavy chain constant regions comprises an amino acid modification (e.g. M84C) which promotes the multimerisation or polymerisation of the fusion protein. Preferably the tailpiece is human and is derived from IgM or IgA and has a substitution or deletion at residue C17. Preferably, each chimeric chain comprises one or more murine hinge regions and a targeting moiety such as an ntigen binding region, small molecule or nucleic acid. The invention further relates to the uses of the fusion protein to detect a target molecule in a sample, preferably as a means for diagnosis such as blood group haemagglutination.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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MURINE ANTIBODIES

FIELD OF THE INVENTION

The present invention relates to a fusion protein. It also relates to a method of detecting the presence of a target molecule in a sample, as well as a composition comprising a fusion protein of the invention.

BACKGROUND

IgM antibodies, as a consequence of their ability to form multimeric structures, are typically used in the antibody diagnostic field due to their enhanced sensitivity and high avidity binding to antigens. For example, mouse IgM antibodies that recognise human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing.

However, IgM antibodies suffer from a number of significant drawbacks that reduce their commercial attractiveness. These antibodies are much less stable than IgG antibodies, and their production is demanding and inefficient. Furthermore, IgM antibodies bind antigens over a narrow pH range and are very sensitive to elevated temperatures and freezing, making them unattractive for use in the developing world. For example, they rapidly lose their agglutination capability after long-term storage or at 42°C, an effect that has been correlated with aggregation and proteolytic trimming of IgM heavy chains.

Consequently, there is need for improved antibodies, which will aim to overcome or ameliorate at least some of the problems associated with the antibodies of the prior art.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a fusion protein comprising two chimeric polypeptide chains, wherein each chimeric polypeptide chain comprises:

• two immunoglobulin G (IgG) heavy chain constant regions, each derived from a mouse IgG 1 heavy chain constant region, and • an immunoglobulin tailpiece derived from a non-native immunoglobulin, and wherein the amino acid sequence of each of the IgG heavy chain constant regions comprise an amino acid modification which promotes the polymerisation of the fusion protein.

In a second aspect the invention provides a method of detecting the presence of a target molecule in a sample, the method comprising the steps of:

• contacting the sample with a fusion protein of the first aspect, • assaying binding of a target molecule to the fusion protein of the first aspect, and • thereby detecting the presence of a target molecule in the sample.

In a third aspect the invention provides a composition comprising fusion proteins of the first aspect of the invention, wherein at least 30% of the fusion proteins in the composition are present in the form of polymeric structures comprising at least 6 fusion proteins.

It will be appreciated that the polymeric structures present in the compositions of the third aspect are formed as a result of interactions between the individual fusion proteins. Such interactions are mediated by the amino acid modifications which promote polymerisation of the fusion protein. Examples of suitable amino acid modifications are contemplated elsewhere in this specification.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of DNA constructs of mouse fusion proteins forming monomers and DNA constructs of exemplary proteins of the invention.

Figure 2 shows the results of Western blotting, illustrating the production of polymers by fusion proteins of the invention.

Figure 3 shows results of size-exclusion chromatography illustrating the native molecular weights of different mouse Fc proteins, including the hexameric mouse Fc.

Figure 4 shows the results of Western blotting illustrating that an antigen can be fused to mouse lgG1 containing a single hinge (sequence 12) or the five-hinge moiety (sequence 13)

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the inventor’s development of a novel fusion protein.

The fusion protein is principally derived from a modified murine immunoglobulin G1 (lgG1) heavy chain constant region and a non-immunoglobulin G derived tailpiece. The combination of a murine IgG 1 sequence with specific amino acid modifications enables the fusion protein to form polymeric structures. Such structures are able to carry an increased number of target molecule binding sites and potentially have greater avidity for binding of target molecules. Both of these properties are highly desirable in the context of a diagnostic tool.

It will be appreciated that testing of human samples is of particular clinical and commercial interest. As such, the use of a murine amino acid sequence as a starting point for the fusion protein of the invention is beneficial. Proteins comprising such a sequence may be less likely to interact with auto-antibodies, for example human auto-antibodies, which may be present in a tested sample. The interaction of auto-antibodies with the fusion protein could potentially lead to an incorrect diagnosis, for example caused by a false positive result. Therefore, the reduction of the likelihood that the fusion protein of the invention will react with such autoantibodies renders the fusion protein useful in providing a more robust diagnostic tool, enabling the development of assays with greater specificity.

The inventors previously described therapeutic fusion protein (WO/2014/060712) is also capable of polymerising into multimeric structures (known as Hexa-Fc). The fusion proteins of Hexa-Fc comprise a region derived from human lgG1, a tailpiece derived from human IgM, and a modification in the lgG1 amino acid sequence. Hexa-Fc was shown to be of therapeutic utility, especially in the context of IVIG, where the protein’s binding to Fey receptors is generally highly desirable.

The structure of the fusion protein of the invention, whilst may resemble the structure of the inventor’s previously developed human derived fusion protein, comprises a surprising difference. It is this difference that enables the formation of the desirable polymeric structures obtained from a fusion protein comprising mainly a murine amino acid sequence.

The inventor’s attempt at making a murine form of Hexa-Fc failed when a murine fragment derived from lgG2a (which most closely resembles the human IgG 1) was used. Such fusion proteins were unable to form polymeric structures. However, the inventors found that the formation of such polymeric structures is possible when a murine lgG1 sequence accompanied by specific amino acid modifications is employed instead of lgG2a.

This finding was particularly surprising given the high degree of sequence identity between murine lgG2a and IgG 1, and the fact that murine lgG2a (but not IgG 1) shares many of the same characteristics as human IgG 1 (such as binding the same Fey receptors). Therefore, the inventors had strong reason to believe that since fusion proteins comprising murine lgG2a did not polymerise, fusion proteins comprising IgG 1 would also certainly not do so.

The following definitions may be useful in further describing and understanding the invention.

Fusion protein

For the purposes of the present invention, a fusion protein may be considered to be any non-natural protein composed of portions of at least two different naturally occurring proteins. Suitably a fusion protein of the invention may comprise portions of at least three, four, or more different proteins.

Suitably, a fusion protein of the invention comprises chimeric polypeptide chains having a length of at least 50, at least 60, at least 70, least 80, least 90, least 100, least 110, least 120, least 130, least 140, least 150, least 160, least 170, least 180, least 190, least 200, least 210, least 220, at least 230, or at least 240 amino acid residues.

Suitably, a fusion protein of the invention comprises chimeric polypeptide chains having a length of up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180, up to 170, up to 160, up to 150, up to 140, up to 130, up to 120, up to 110, up to 100, up to 90, up to 80, up to 70, up to 60 or up to 50, amino acid residues.

Suitably, a fusion protein of the invention comprises chimeric polypeptide chains having a length of 244 amino acid residues. The amino acid sequence of the chimeric polypeptide chains of such an exemplary fusion protein of the invention is set out in SEQ ID NO: 16 or SEQ ID NO: 18.

Merely by way of example, and as long as the requirements of the various aspects of the invention are met, a fusion protein of the invention may comprise chimeric polypeptide chains sharing at least 80% identity with SEQ ID NO: 16 or SEQ ID NO: 18. Suitably a fusion protein of the invention may comprise chimeric polypeptide chains sharing at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 16 or SEQ ID NO: 18..

It will be appreciated that a fusion protein of the invention may be formed of chimeric polypeptide chains comprising SEQ ID NO: 16 or SEQ ID NO: 18. Alternatively, a fusion protein of the invention may be formed of chimeric polypeptide chains consisting of SEQ ID NO: 16 or SEQ ID NO: 18.

Amino acid modifications

The fusion proteins of the invention are based upon naturally occurring immunoglobulin sequences, as described in detail elsewhere in the specification.

Generally, suitable modifications may be selected from the group consisting of: substitution of an amino acid with a non-native amino acid, deletion of an amino acid, or addition of a non-native amino acid. In this context a “non-native” amino acid is an amino acid residue that is not the same as the amino acid residue found at the corresponding position in the native “wild-type” protein. “Corresponding” amino acid residues are considered in more detail below.

In embodiments in which more than one modification is present, the different modifications may be independently selected from the group consisting of: amino acid substitutions; amino acid deletions; and amino acid additions.

“Corresponding” amino acids

The present invention makes use of peptide sequences based on those found in naturally occurring molecules, particularly those found in naturally occurring immunoglobulin proteins. In keeping with the chimeric nature of the proteins of the invention, sections of these naturally occurring sequences are fused with one another, and with modified portions of naturally occurring sequences, to produce the fusion proteins of the invention.

It will be appreciated that the modifications made, and the optional truncations, fusions, and rearrangement of the immunoglobulin sequences, mean that, while certain biologically relevant motifs of amino acid residues from the natural molecules are retained in the fusion proteins of the invention, the numbering of individual amino acid residues will not be the same as in the original natural protein. That said, the skilled person will understand that the constituents and arrangement of the amino acid residues in these motifs remain recognisable in the fusion proteins of the invention when compared the naturally occurring sequences from which they are derived. Thus, the amino acid residue in a fusion protein of the invention corresponds to the same amino acid residue in a naturally occurring molecule, even if its’ position in the molecule as a whole has changed.

In the context of the present invention, references to a “corresponding amino acid” in a fusion protein of the invention should be construed accordingly. The skilled person will be able to identify an amino acid residue in a fusion protein of the invention as corresponding to one from a naturally occurring sequence if it has the same position within a motif or contiguous sequence of residues from a naturally occurring protein, and/or if it serves the same biological role in the fusion protein of the invention as in the naturally occurring protein.

Mouse lgG1 heavy chain constant region

The mouse IgG 1 heavy chain constant region is one of the naturally occurring sequences on which a part of the fusion proteins of the invention is based. As noted above, the heavy chain constant regions of the fusion proteins of the invention are “derived from” the mouse lgG1 sequence. The versions in the fusion proteins of the invention are not themselves the naturally occurring sequences, but sequences that have been modified at least to promote the polymerisation of fusion proteins of the invention. Both immunoglobulin heavy chain constant regions of a fusion protein of the invention incorporate such modifications. Suitably each heavy chain constant region comprises the same modifications.

The amino acid sequence of the mouse lgG1 heavy chain constant region is well known to those skilled in the art. For the avoidance of doubt, an exemplary wild type mouse lgG1 heavy chain constant region sequence for is set out in SEQ ID NO: 2. The sequence of SEQ ID NO: 2 may be used as a reference to determine the presence of absence of amino acid modifications in a mouse IgG 1 heavy chain constant region, such as one incorporated in a fusion protein of the invention.

As referred to above, the portion of the IgG heavy chain constant region incorporated in a fusion protein of the invention comprises an amino acid modification which promotes the polymerisation of the fusion protein. In particular, the modification may comprise a modification of the methionine residue at position 84 (M84) of SEQ ID NO: 2.

A suitable modification which promotes the polymerisation of the fusion protein may be a substitution of an amino acid, such as M84 with a non-native amino acid. Alternatively a suitable modification which promotes the polymerisation of the fusion protein may be a deletion of an amino acid, such as M84. Still further, a suitable modification which promotes the polymerisation of the fusion protein may be an addition of a non-native amino acid.

In a suitable embodiment, the modification that promotes polymerisation of the fusion protein is substitution of M84 with a non-native amino acid. In a particularly suitable embodiment, the substitution of M84 is with a cysteine residue. Modification of the mouse lgG1 heavy chain constant region to incorporate a M84C substitution is set out in SEQ ID NO: 3.

It will be appreciated that, in embodiments in which more than one modification which promotes the polymerisation of the fusion protein is present, the different modifications may be independently selected from the group consisting of: amino acid substitutions; amino acid additions; and amino acid deletions.

The introduction of one, or more, further cysteine residues within the IgG heavy chain constant region provides a suitable modification of this region which promotes the polymerisation of the fusion proteins of the invention. The additional cysteine residues may be introduced as modifications (replacing residues found in the native sequence, as with the substitution of M84 considered above) or may be introduced as additional amino acid residues further to the native sequence already present.

The modifications considered above are merely exemplary, and do not constitute a definitive list of suitable modifications of this sort. As mentioned above, the skilled person will readily be able to determine whether or not a modification of the native sequence is present. Methods by which the ability of such a modification to promote polymerisation may be assessed are considered below.

Suitably the portion of the IgG heavy chain constant region incorporated in a fusion protein of the invention may comprise more than one amino acid modification which promotes the polymerisation of the fusion protein. For example, the portion of the IgG heavy chain constant region may comprise 2 or more such modifications, 3 or more such modifications, 4 or more such modifications, or 5 or more such modifications. Indeed, the portion of the IgG heavy chain constant region may comprise 6 or more such modifications, 6 or more such modifications, 8 or more such modifications, 9 or more such modifications, or 10 or more such modifications.

The portion of the IgG heavy chain constant region incorporated in a fusion protein in accordance with the invention may comprise no more than 10 amino acid modifications, no more than 9 modifications, no more than 8 modifications, no more than 7 modifications, no more than 6 modifications, no more than 5 modifications, no more than 4 modifications, no more than 3 modifications, or no more than 2 amino acid modifications as compared to the reference sequence. An IgG heavy chain constant region incorporated in a fusion protein of the invention may comprise a single amino acid modification as compared to a wild type sequence.

Suitably an IgG heavy chain constant region incorporated in a fusion protein of the invention may share at least 80% identity with the reference sequence of SEQ ID NO: 2. Suitably an IgG heavy chain constant region incorporated in a fusion protein of the invention may share at least 85% identity, at least 90% identity, or at least 95% identity with the reference sequence of SEQ ID NO: 2. Merely by way of example, and as long as the requirements of the various aspects of the invention are met, a fusion protein of the invention may comprise chimeric polypeptide chains sharing at least 80% identity with SEQ ID NO: 2. Suitably a fusion protein of the invention may comprise chimeric polypeptide chains sharing at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 2.

Each fusion protein of the invention comprises two heavy chain constant regions derived from mouse lgG1. Both heavy chain constant regions present in a fusion protein of the invention may be identical. Alternatively a fusion protein of the invention may comprise two different heavy chain constant regions derived from mouse lgG1.

A heavy chain constant region incorporated in a fusion protein of the invention may have a length of at least 50, at least 60, at least 70, least 80, least 90, least 100, least 110, least 120, least 130, least 140, least 150, least 160, least 170, least 180, least 190, least 200, least 210, or least 220 amino acid residues.

A heavy chain constant region incorporated in a fusion protein of the invention may have a length of up to 230, up to 220, up to 210, up to 200, up to 190, up to 180, up to 170, up to 160, up to 150, up to 140, up to 130, up to 120, up to 110, up to 100, up to 90, up to 80, up to 70, up to 60 or up to 50, amino acid residues.

Suitably a fusion protein of the invention comprises a heavy chain constant region having length of 244 amino acid residues. An exemplary amino acid sequence of a heavy chain constant region of the protein of the invention is set out in SEQ ID NO: 2.

It will be appreciated that, in embodiments in which only a portion of the reference wild-type IgG heavy chain constant region is present in a fusion protein of the invention, references to comparison with the reference sequence should be construed accordingly.

An immunoglobulin tailpiece from, or derived from, a non-native immunoglobulin

The fusion proteins of the invention comprise an immunoglobulin tailpiece from, or derived from, a non-native immunoglobulin. In the present context, the term non-native is intended to refer to the relationship between the immunoglobulin tailpiece and the IgG heavy chain constant region derived form a mouse lgG1 heavy chain constant region. Thus the nonnative tailpiece is the tailpiece from an immunoglobulin other than mouse IgG 1.

In the context of the immunoglobulin tailpiece (but not the mouse IgG 1 heavy chain constant region), references to a tailpiece “from” a non-native immunoglobulin should be taken as directed to sequences corresponding to the naturally occurring non-native tailpiece. On the other hand, references to a tailpiece “derived from” a non-native immunoglobulin should be taken as directed to modified sequences based upon such naturally occurring non-native tailpieces.

Suitably the non-native immunoglobulin tailpiece may be from, or derived from, an immunoglobulin G other than lgG1. Suitably the non-native immunoglobulin tailpiece may be from, or derived from, an immunoglobulin other than IgG.

Suitably the immunoglobulin tailpiece is from, or derived from, an immunoglobulin selected from the group consisting of: IgA; IgM

Suitably non-native immunoglobulin tailpiece may be derived from, or derived from, a species other than mouse. Since the IgG heavy chain constant region employed in the proteins of the invention is murine in origin, in such embodiments, the fusion protein of the invention is composed of at least two different proteins from at least two different species.

In a suitable embodiment, the immunoglobulin tailpiece is from, or derived from, a human immunoglobulin. The immunoglobulin tailpiece may be from, or derived from, human IgA, or human IgM.

Sequences derived from an immunoglobulin tailpiece

Those immunoglobulin tailpieces incorporated in a fusion protein of the invention that are derived from a naturally occurring protein are modified as compared to the wild type tailpiece from which they are derived. The amino acid sequences of the tailpieces of IgA and IgM are well known to those skilled in the art. For the avoidance of doubt, an exemplary wild type sequence of the tailpiece of human IgA is set out in SEQ ID NO: 4, and an exemplary wild type sequence of the tailpiece of human IgM is set out in SEQ ID NO: 5. Either SEQ ID NO: 4 or SEQ ID NO: 5 are suitable for use as non-native immunoglobulin tailpiece sequences in the fusion proteins of the invention.

The sequences of SEQ ID NOs: 4 and 5 may be used as a reference to determine the presence or absence of amino acid modifications in tailpieces incorporated in a fusion protein of the invention.

In the case of a fusion protein of the invention comprising an immunoglobulin tailpiece derived from a naturally occurring protein, the tailpiece may share at least 80% identity with the immunoglobulin tailpiece region of SEQ ID NO: 4 or 5. Suitably a fusion protein of the invention may comprise an immunoglobulin tailpiece sharing at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the immunoglobulin tailpiece region of SEQ ID NO: 4 or 5.

A fusion protein in accordance with the invention may comprise a tailpiece that comprises one or more modifications that promote polymerisation of the fusion protein. Methods by which the ability of a modification to promote polymerisation may be assessed are considered elsewhere in this speciation.

In a suitable embodiment, a fusion protein of the invention may comprise a tailpiece comprising a modification of the amino acid residue corresponding to C575 of IgM (residue 17 of SEQ ID NO: 5) orC471 of IgA (residue 17 of SEQ ID NO: 4). Suitably the modification may be a substitution or deletion of the residue corresponding to C575 of IgM or C471 of IgA. Suitably a fusion protein of the invention comprises a non-native immunoglobulin tailpiece that comprises a substitution of the residue corresponding to C575 with alanine.

Modifications of this sort promote the formation of monomeric fusion proteins of the invention which comprises a glycan molecule. Such glycosylated monomeric fusion proteins of the invention may also be referred to as “complex monomers”. Fusion proteins of the invention in accordance with this embodiment may also aggregate to form laddered polymers of the fusion proteins.

Suitably, the complex monomer is obtained as a result of a substitution of a tailpiece cysteine residue with a non-native amino acid. Suitably, the cysteine is substituted with alanine. Suitably the substitution is at a position corresponding to C575 of IgM or C471 of IgA.

The inventors believe that such a substitution may render a glycan molecule available for functional interactions. By way of example, when the residue corresponding to C575 of IgM is substituted with alanine, a glycan molecule at position N231 of SEQ ID NO: 18 may be free to interact with receptors. In the absence of the C575A substitution, the glycan molecule at position N231 of SEQ ID NO: 18 would not be free for such an interaction. It will be appreciated that residue N231 of SEQ ID NO: 18 corresponds to residue amino acid 5 of SEQ ID NO: 4 or SEQ ID NO: 5, and also corresponds to amino acid 563 of human IgM or amino acid 459 of human IgA.

The term “laddered polymers” as used herein refers to a polymer formed by complex monomers which are able to form disulphide bonds between individual complex monomers, thereby forming a laddered effect. By way of example a laddered polymer may be formed by complex monomers having M84C modification (SEQ ID NO: 3).

Additionally, or alternatively, a fusion protein of the invention may comprise a modification of the amino acid residue corresponding to N563 of IgM (amino acid residue 5 of SEQ ID NO: 5) or a modification of the amino acid residue corresponding to N459 of IgA (residue 5 of SEQ ID NO: 4). Suitably the modification may be a substitution or deletion of the residue corresponding to N563 of IgM or of N459 IgA. Such modifications cause the disruption of a glycosylation site in the fusion proteins of the invention.

In a suitable embodiment, a fusion protein of the invention comprises a non-native immunoglobulin tailpiece that comprises a substitution of the residue corresponding to N563 of IgM with alanine or that comprises a substitution of the residue corresponding to C575 of IgM with alanine. Modifications of the amino acid residue corresponding to N563 of IgM or N459 of IgA promote polymerisation of the fusion proteins in which they are present, and may promote the formation of dodecamers.

A tailpiece incorporated in a fusion protein of the invention may comprise a single modification. Alternatively, a suitable tailpiece may comprise at least 2 modifications, at least 3 modifications, at least 4 modifications, at least 5 modifications, at least 6 modifications, at least 7 modifications, at least 8 modifications, at least 9 modifications, or at least 10 modifications.

As before, suitable modifications include substitution of an amino acid, deletion of an amino acid, or addition of a non-native amino acid. In embodiments in which more than one modification is present, the different modifications may be independently selected from the group consisting of: amino acid substitutions; amino acid additions; and amino acid deletions.

A hinge region

Antibody heavy chains comprise a hinge region, located between the constant and variable regions. The sequences of such hinge regions are well known to those skilled in the art.

Such hinge regions may also be present in the fusion proteins of the invention. Merely by way of example, the sequence GCKPCICT (SEQ ID NO: 12) corresponds to the hinge sequence of murine IgG 1. Hinge regions of IgG, and particularly of IgG 1, or modified forms of such hinge regions, are suitable for use in the fusion proteins of the invention.

A fusion protein in accordance with the present invention may comprise a naturally occurring hinge region. Alternatively, or additionally, a fusion protein in accordance with the present invention may comprise a modified hinge region. Suitably a fusion protein in accordance with the present invention comprises a plurality of hinge regions. The hinge regions are suitably located at the C-terminus of the domain derived from the heavy chain constant region of mouse IgG 1.

The presence of a plurality of hinge regions in a fusion protein of the invention may confer many benefits, as discussed below.

Suitably a fusion polypeptide of the invention may comprise a payload moiety. The term “payload moiety” is defined elsewhere in this specification. In an embodiment where the fusion polypeptide comprises a payload moiety, the plurality of hinge regions may be located between the payload moiety and the C-terminus of the domain derived from an immunoglobulin heavy chain constant region. The presence of a payload moiety may be desirable, for example, for research purposes or therapeutic purposes, as considered below.

Suitably a fusion polypeptide of the invention may comprise a targeting moiety. The term “targeting moiety” is defined elsewhere in this specification. In an embodiment where the fusion polypeptide comprises a targeting moiety, the plurality of hinge regions may be located between the targeting moiety and the C-terminus of the domain derived from an immunoglobulin heavy chain constant region. The presence of a targeting moiety may be desirable, for example, for testing immune responses in animal models, such as mouse models.

In the case of a fusion protein of the invention comprising a plurality of hinge regions, the plurality of hinge regions may increase the distance between the domain derived from the heavy chain constant region of mouse lgG1 and the payload or targeting moiety, if such moiety is present. Increased distance between the domain and the moiety may enable the binding of the domain to FcRs (for example Fey Ila and Fey Illa). The inventors believe that the plurality of hinge regions may provide sufficient space and flexibility between the domain and the moiety to allow the attachment of a glycan molecule to a glycosylation site on the fusion polypeptides or proteins of the invention. The presence of a glycan molecule may enable the polypeptides and proteins of the invention to bind FcRs and/or glycan receptors. The inventors also believe that the location of the glycosylation site may influence whether the fusion polypeptides and proteins of the invention bind FcRs, glycan receptors (for example sialic acid receptors such as SIGLEC-1), or both. Suitable glycosylation sites, and the ways in which they may influence the binding of the polypeptides and proteins of the invention, are discussed elsewhere in this specification.

In a suitable embodiment a hinge region may be at least 4, at least 5, at least 6, at least 7, least 8, at least 9, at least 10, at least 11, least 12, at least 13, at least 14, at least 15, least

16, at least 17, at least 18, at least 19, at least 20, or more, amino acids long. Suitably, the hinge region is at least 8 amino acids long.

In a suitable embodiment a hinge region may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, or more, amino acids long. Suitably, the hinge region is 8 amino acids long.

In a suitable embodiment, a fusion protein of the invention comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more hinge regions.

Suitably, the fusion protein of the invention comprises at least 2 hinge regions. Suitably, the protein of the invention comprises at least 3 hinge regions. More suitably, the protein comprises at least 4 hinge regions. Most suitably the protein comprises at least 5 hinge regions.

In a suitable embodiment, the fusion protein of the invention comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hinge regions. Suitably, the fusion protein of the invention comprises 2 hinge regions. Suitably, the fusion protein of the invention comprises 3 hinge regions. More suitably, the fusion protein of the invention comprises 4 hinge regions. Most suitably, the fusion protein comprises 5 hinge regions.

An example of a sequence comprising a plurality of hinge regions suitable for use in the fusion protein of the invention is set out in SEQ ID NO: 13. This sequence comprises 5 hinge regions.

In a suitable embodiment one or more, or all of the plurality of hinge regions may be separated from each other by a short amino acid sequence.

Suitably, the short amino acid sequence may be up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 amino acid residues in length. Suitably, one or more, or all, of the hinge regions may be separated from one another by short amino acid sequences of 2 amino acid residues.

Suitably, the short amino acid sequence may be selected from the group consisting of RS, SR, and VD. It will be appreciated that when more than two hinge regions are present within the hinge area, several hinge regions may be separated by different short amino acid sequence, while others not separated by a short amino acid sequence at all. For example, in an embodiment where the fusion protein comprises five hinge regions, the first and second hinge regions may be separated by the amino acid sequence SR, the second and third hinge regions may be separated by the amino acid sequence VD, and the third and fourth hinge regions may be not separated by a short amino acid sequence, and the fourth and fifth hinge region may be separated by an amino acid RS.

In a suitable embodiment the short amino acid sequence may be a restriction site. The presence of a restriction site may enable or simplify the addition and/or removal of one or more hinge regions to the fusion protein of the invention.

The inventors believe that the presence of a short amino acid sequence between the hinge regions may confer a number of additional advantages. For example, the short amino acid sequence may allow for the insertion of a glycan sequon without interfering with the structure of the hinge region(s). Suitably, the glycan sequon may be according to SEQ ID NO: 39 (N-X-S/T; in which N represents an asparagine residue, X represents an amino acid residue other than proline or asparagine, and S/T represents a serine or threonine residue).

Additionally or alternatively, the short amino acid sequence may render the hinge region(s) resistant to cleavage by a number of proteases. Suitably, in such an embodiment, the short amino acid sequences may disrupt restriction sites that would otherwise be present.

Surprisingly, the inventors have found that the short amino acid sequence may confer the above mentioned advantages without adversely affecting the ability of the fusion protein of the invention to bind a Fc receptor, even in the presence of a targeting moiety.

In a fusion protein of the invention where the hinge regions contain one or more cysteine residues, the hinge regions of the first fusion polypeptide may bind the hinge regions of the second fusion polypeptide. Such binding may be via an inter-disulphide bond. It will be appreciated that when the hinge regions of at least one of the first and/or second fusion polypeptide forming the monomeric protein lacks cysteine residues such binding may be prevented. Accordingly, a fusion protein in accordance with the invention may comprise one or more hinge regions that lack cysteine residues. As cysteine residues are a common feature of naturally occurring hinge regions, such cysteine-lacking hinge regions may be modified forms of naturally occurring hinge regions.

Cysteines in hinge region sequences may be substituted by any appropriate amino acid residue to provide a hinge region that lacks cysteine residues. For example, the sequence GAKPAIAT (SEQ ID NO: 14) corresponds to a cysteine-free version of the hinge sequence of murine lgG1.

It will be appreciated that a fusion protein of the invention comprising a plurality of hinge regions may comprise solely hinge regions that comprise cysteine residues. Alternatively, a fusion protein of the invention comprising a plurality of hinge regions may comprise solely hinge regions that lack cysteine residues. Suitably a fusion protein of the invention with a plurality of hinge regions comprises a combination of hinge regions that comprise cysteine residues, and hinge regions that lack cysteine residues.

The inventors believe that the removal of a cysteine residue(s) in one or more hinge regions is advantageous as it may increase the flexibility of the hinge regions and thereby impart greater flexibility between the domain derived from the heavy chain constant region, and the payload or targeting moiety, if present. This in turn, may enable the use of larger moieties and/or increase the reach of moieties to their ligands. Furthermore, the inventors also believe that hinge regions with reduced or no cysteine residues may be more resistant to cleavage by a number of proteases, which cleave peptides near cysteine residues.

It will be appreciated that hinge region flexibility may be particularly desirable in at least one of the hinge regions which are adjacent to the payload or targeting moiety. Merely by way of example, in a fusion protein of the invention which comprises four hinge regions, the hinge region closest to the domain derived from an immunoglobulin heavy chain consent region may contain cysteine residues, while the remaining three hinge regions do not. Alternatively, in a fusion protein of the invention which comprises four hinge regions, the two hinge regions closest to the domain derived from an immunoglobulin heavy chain consent region may contain cysteine residues, while the remaining two hinge regions do not.

A suitable sequence providing a plurality of hinge regions comprising a combination of cysteine-containing and non-cysteine-containing hinge regions that may be incorporated in a fusion protein of the invention is as follows:

GAKPAIATSRGAKPAIATVDGAKPAIATGAKPAIATRSGCKPCICT (SEQ ID NO: 15).

A targeting moiety

A fusion protein in accordance with the invention may further comprise a targeting moiety. A suitable targeting moiety is able to confer specificity of binding to a fusion protein of the invention.

Suitably the targeting moiety may enable specific binding of the fusion protein of the invention to a target molecule of interest. Target molecules of interest are discussed elsewhere in this disclosure (in particular in the context of the methods of the invention).

Merely by way of example, and without limitation, a suitable targeting moiety for incorporation in a fusion protein of the invention may be selected from the group consisting of: an antigen, or antibody-binding portion thereof; an antibody, or antigen-binding portion thereof; an affimer; an aptamer; an adhiron; a DNA molecule; an RNA molecule; a protein; and a small molecule.

Embodiments in which an antigen, or antibody-binding fragment thereof, is used as the targeting moiety are particularly suitable for the detection of antibody target molecules. It will be appreciated that such embodiments may be beneficial in the context of use of the fusion proteins of the invention to bind to autoantibodies associated with autoimmune diseases. Such uses may include diagnostic or prognostic applications. In a suitable embodiment, a targeting moiety may comprise an epitope associated with an autoimmune disease.

Embodiments in which antibodies, or their fragments, affimers, aptamers, or adhirons are used as the targeting moiety may be used in any case in which it is desired to target a target molecule capable of being bound by such moieties. An scFv fragment represents a suitable example of an antigen-binding portion of an antibody that may be incorporated as a targeting moiety in a fusion protein of the invention.

The targeting moiety may be associated with the fusion protein of the invention by any suitable means that allow the targeting moiety to confer specificity of binding. Suitably, the targeting moiety may be associated with the protein of the invention by one or more hinge regions.

In the case of a protein targeting moiety, the targeting moiety may be fused with the fusion protein of the invention. In the case of a non-protein targeting moiety, the targeting moiety may be conjugated to the fusion protein, for example by covalent bonding, or by weak interactions.

It will be appreciated that, the specificity of binding conferred by the targeting moiety may be considered in determining the suitability of a fusion protein of the invention for use in a method of the invention. In particular, a fusion protein having a targeting moiety that confers specific binding for a particular target molecule of interest can be used to bind to that target of interest in a method of the invention. Merely by way of example, a targeting moiety that confers specific binding for CD 19 may be used in a fusion protein of the invention that is to be employed in a method of the invention directed to detecting the presence of CD19 in a sample.

A payload moiety

As touched upon above, in a suitable embodiment, the fusion protein of the present invention may comprise a payload moiety. Examples of suitable payload moieties are described below.

The payload moiety may be naturally occurring or synthetic. A naturally occurring payload moiety is one that can be found in nature. A synthetic payload moiety is one that does not exist in nature (for example one that is manmade).

In a suitable embodiment, a naturally occurring payload moiety is a proteinaceous molecule, or a non-proteinaceous molecule.

In a suitable embodiment, a naturally occurring or synthetic payload moiety may be selected from the group consisting of a chemical compound, a proteinaceous molecule, a nucleic acid molecule, a lipid, and a carbohydrate. It will be appreciated that a synthetic proteinaceous molecule or a synthetic nucleic acid molecule may have a sequence which is derived from a naturally occurring sequence.

A proteinaceous molecule may be selected from the group consisting of a protein, a peptide and a peptidomimetic. By way of example, a proteinaceous molecule may be an antigen, a pathogen-associated molecular pattern (PAMP), a ligand, a receptor, a cytokine or a chemokine.

A non-proteinaceous molecule may be selected, for example, from the group consisting of a chemical compound, a nucleic acid molecule, a lipid and a carbohydrate. Other nonproteinaceous molecules will be known to the skilled person.

In a suitable embodiment, a chemical compound may be a small molecule. By way of example and not limitation, suitable small molecules may be selected from the group consisting of mertansine, monomethylauristatin E, doxorubicin and N-acetyl-γ calicheamicin.

In a suitable embodiment, the payload moiety may be fused to the fusion polypeptide or protein of the invention. Alternatively, the payload moiety may be conjugated with the polypeptide or protein of the invention.

In an embodiment where the payload moiety is fused to the fusion protein of the invention, the amino acid sequence encoding the payload moiety is located within the same reading frame as the amino acid sequence of the polypeptide of the invention. The fused payload moiety may thus be expressed as part of the same gene product as the fusion protein. As such, it will be appreciated that a payload moiety that is fused to the fusion polypeptide of the invention is a proteinaceous molecule. Such a fused proteinaceous molecule may be naturally occurring or synthetic.

The term “conjugated” as used herein refers to an interaction between a fusion polypeptide or protein of the invention and a non-proteinaceous molecule by means of covalent bonding, or by means of weak interactions.

In a suitable embodiment, the payload moiety may be selected from the group consisting of: a therapeutic agent; a diagnostic agent; and a research agent. Each of these may be naturally occurring or synthetic.

In a suitable embodiment, a therapeutic agent is a molecule which has a therapeutic effect. Such a therapeutic effect may include amelioration of a symptom and/or disease, delay of onset of a symptom and/or disease, and/or prevention of onset of a symptom and/or disease. The therapeutic effect of a therapeutic agent may be in addition to, or independent of, the therapeutic effect of the polypeptide or protein of the invention. Such an embodiment of the fusion protein of the invention may have a particular application in veterinary medicine.

A therapeutic agent may be selected from the group consisting of a drug, a carbohydrate, a nucleic acid and a proteinaceous molecule.

The term “drug” as used herein refers to a chemical compound with therapeutic activity, for example a small molecule, which may be conjugated to a fusion polypeptide or protein of the invention. Merely by way of example, a suitable drug therapeutic molecule 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 fusion protein of the invention may be conjugated to an anti-cancer drug, such as monomethyl auristatin E.

Merely by way of example, a suitable proteinaceous molecule may be a protein, such as 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.

In another example, the proteinaceous molecule is a protein which is an immune modulator. A fusion protein of the invention fused or 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 pathogenassociated molecular pattern (PAMP) molecule or an antigen.

A fusion protein of the invention fused or 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 example 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 or fused to a fusion protein of the invention. Such a conjugated protein may be used in the prevention or treatment of an autoimmune disease.

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

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

In a suitable embodiment, a diagnostic agent is a molecule or compound which may detect the presence of a target molecule within the subject and/or a test sample. The presence of a target molecule may be indicative of a disease. Suitably, the diagnostic agent may be for use in in vivo and/or in vitro diagnosis. More suitably it may be for use in in vitro diagnosis. Suitably it may be not for use in in vivo diagnosis.

An amino acid modification which promotes the polymerisation of the fusion proteins

Examples of amino acid modifications which promotes the polymerisation of the fusion proteins of the invention have been described above. These include examples of such modifications that may be incorporated in the sequence from the IgG heavy chain constant region, as well as examples of such modifications that may be incorporated in the sequence from the Ig tailpiece.

For the avoidance of doubt, in the terms of the present disclosure, an “amino acid modification which promotes the polymerisation” of fusion proteins of the invention may be one that increases the proportion of fusion proteins that polymerise. Alternatively or additionally, an “amino acid modification which promotes the polymerisation” of fusion proteins of the invention may be one that increases the average number of fusion proteins of the invention incorporated in a polymeric structure.

The inventors have found that the fusion proteins of the invention are capable of polymerising to produce hexamers, a degree of polymerisation that they believe has not previously been able to be achieved using proteins based upon mouse immunoglobulin sequences.

In the context of the present disclosure, a “hexamer” is a polymeric structure made up of 6 fusion proteins, which in turn are made up of 12 fusion polypeptide chains. The fusion proteins of the invention are capable of assembling into polymeric structures comprising more than 6 fusion proteins, including, but not limited to, those selected from the group consisting of: heptamers (comprising 7 fusion proteins, a total of 14 fusion polypeptide chains); octamers (comprising 8 fusion proteins, a total of 16 fusion polypeptide chains); nonamers (comprising 9 fusion proteins, a total of 18 fusion polypeptide chains); decamers (comprising 10 fusion proteins, a total of 20 fusion polypeptide chains); undecamers (comprising 11 fusion proteins, a total of 22 fusion polypeptide chains); and dodecamers (comprising 12 fusion proteins, a total of 24 fusion polypeptide chains).

Further modifications

A fusion protein of the invention may further comprise an additional N-linked glycosylation site not found in the naturally occurring immunoglobulin sequences from which it is derived.

Suitably such a site may comprise the sequence: N-X-S/T in which N represents an asparagine residue, X represents an amino acid residue other than proline or asparagine, and S/T represents a serine or threonine residue.

The introduction of an additional N-linked glycosylation site in this manner may confer a number of advantages on a fusion protein of the invention. Merely by way of example, the inventors believe that such fusion proteins may benefit from one or more of the following advantages: improved solubility; improved expression; and improved binding to protein G. It will be appreciated that improved solubility may be advantageous in the manufacture or formulation of the fusion proteins of the invention. Improved expression may be expected to increase the yield of a fusion protein of the invention incorporating this modification, as compared to a fusion protein of the invention in which the additional N-linked glycosylation site is not present. Improved binding to protein G may be expected to facilitate the purification of the fusion proteins in which such an additional N-linked glycosylation site is found.

Suitably an additional N-linked glycosylation site may be introduced within a hinge of a fusion protein of the invention, within the heavy chain constant region of such a fusion protein, or within the tailpiece region. SEQ ID NO: 35 and 36 are examples of sequences with an additional N-linked glycosylation site in the hinge region. SEQ ID NO: 37 is an example of a sequence with an additional N-linked glycosylation site in the heavy chain constant region. SEQ ID NO: 38 is an example of a sequence with an additional N-linked glycosylation site in the tailpiece region. However, it will be appreciated that other glycosylation sites, and particularly other N-linked glycosylation sites, may be introduced into the fusion protein of the invention.

Methods of the invention

The methods of the invention allow the detection of the presence of a target molecule in a sample of interest, by employing the fusion proteins of the invention. The sample of interest is contacted with a fusion protein of the invention, and assaying is undertaken to determine binding of a target molecule to the fusion protein. The presence of a target molecule in the sample can thereby be detected.

In a suitable embodiment, the fusion protein of the invention utilized in a method of the invention is one that comprises a targeting moiety that confers on the fusion protein of the invention specificity of binding for the target molecule that the method is directed to detect. Suitable examples of targeting moieties are discussed above.

Fusion proteins of the invention, for use in the methods of the invention, may be provided as part of a panel of fusion proteins that have specificity for different target molecules of potential interest. In such embodiments it may be necessary to distinguish between the different fusion proteins of the invention when assayed, in order to allow such assays to effectively distinguish between the between the different target molecules bound by the fusion proteins. This may be achieved by the use of different reporters in respect of fusion proteins of the invention having different specificities. Assays by which binding of fusion proteins of the invention to target molecules can be determined, and the presence of bound target molecules thus detected, are discussed in more detail below.

Fusion proteins and methods of the invention may be directed to a wide range of target molecules. Merely by way of example, suitable target molecules may be selected from the group consisting of: proteins and nucleic acids. Without limitation, suitable examples of proteins include those selected from the group consisting of: growth factors; antibodies; and cell surface markers. In a suitable embodiment the target molecules are biomarkers.

The nature of the target molecule of interest will inform the decision as to an appropriate targeting moiety to be incorporated in the fusion proteins of the invention. Merely by way of example, in the case of a protein target molecule, a suitable targeting moiety may be an antibody, or antigen binding fragment thereof. In the case of an antibody target molecule, a suitable targeting moiety may be a moiety comprising the epitope bound by the antibody. In the case of a nucleic acid target molecule, a suitable targeting molecule may be a complementary nucleic acid sequence.

The sample may be any sample of interest. For example, a sample may be an environmental sample, or a sample in which it is desired to determine the presence or absence of a desired substance that is being produced (e.g. a product or by product of a reaction). The methods of the invention are particularly suitable for use in the context of clinical sample.

In a suitable embodiment a method of the invention is selected from the group consisting of: a prognostic method, and a diagnostic method. Suitably the method is a diagnostic method, and the method is carried out in vivo.

Methods of the invention may be practiced in respect of any suitable sort of sample. For example, a suitable sample may be selected from the group consisting of: a tissue sample; and a body fluid sample. Samples of this sort are particularly suitable in the context of diagnostic or prognostic methods of the invention.

It will be appreciated that a suitable sample may be selected with reference to the desired outcome of the method of the invention. For example, a suitable sample may be selected with reference to a particular diagnostic or prognostic application.

Without limitation, a suitable tissue sample may be selected from the group consisting of: a muscle tissue sample; a skin tissue sample; a digestive tract tissue sample; a lung tissue sample; a breast tissue sample; a kidney tissue sample; a bone tissue sample; and a neural tissue sample.

In the same manner, a suitable body fluid sample may be selected from the group consisting of: a blood sample; a urine sample; a sputum sample; a bile sample; and a cerebrospinal fluid (CSF) sample.

It will be appreciated that, in the case of diagnostic or prognostic applications of the methods of the invention, the sample may suitably be a biopsy sample.

Methods of the invention may be carried out using samples derived from a wide range of suitable sources. Suitably the sample is a human sample. That said, a suitable sample may be derived from a non-human animal, such a domestic animal, agricultural animal, or experimental animal.

In a suitable embodiment of a method of the invention the target molecule is a biomarker. The biomarker may be associated with a particular diagnostic or prognostic outcome.

Merely by way of example, the biomarker may be indicative of the presence of a disease, disorder or infection. Alternatively, the biomarker may be associated with the likelihood of a desirable or undesirable clinical outcome.

Binding of the fusion protein of the invention to a target molecule may be assayed by any suitable method. The fusion proteins used in the methods of the invention are based upon antibodies, and techniques known from the art of determining antibody binding may be used in the methods of the invention. A suitable assaying technique by which binding of a fusion protein of the invention (and thus the presence of a target molecule of interest) in a sample may be determined, may be selected from the group consisting of: immunofluorescence; enzyme-linked immunosorbent assay (ELISA); surface plasmon resonance (SPR) analysis; and immunoblotting.

Merely by way of example the fusion proteins of the invention may be labelled directly with an appropriate detection moiety. The methods of the invention may employ secondary binding partners, which bind to the fusion proteins of the invention, which are labelled with an appropriate detection moiety. Such secondary binding partners may be used to indirectly label a fusion protein of the invention to which the secondary binding partner has bound.

Suitable examples of secondary binding partners that may be used for such indirect labelling include antibodies that specifically bind to the fusion proteins of the invention.

A suitable detection moiety may comprise a fluorophore (such as TRITC, FITC, or a fluorescent protein), or an enzyme (such as horseradish peroxidase) that can catalyse a colorigenic reaction in the presence of a suitable substrate. Other suitable assays to determine binding of a fusion protein of the invention will be apparent to those skilled in the art.

Compositions

Compositions in accordance with the third aspect of the invention comprise fusion proteins of the invention in a suitable carrier. The carrier is one that permits polymerisation of the fusion proteins of the invention.

As noted in the statements of invention, at least 30% of the fusion proteins in the composition are present in the form of polymeric structures comprising at least 6 fusion proteins. It will be appreciated that in the present context, a “hexamer” is made up of 6 fusion proteins, which in turn are made up of 12 fusion polypeptide chains. The fusion proteins of the invention are capable of assembling into polymeric structures comprising more than 6 fusion proteins, including, but not limited to, those selected from the group consisting of: heptamers (comprising 7 fusion proteins, a total of 14 fusion polypeptide chains); octamers (comprising 8 fusion proteins, a total of 16 fusion polypeptide chains); nonamers (comprising 9 fusion proteins, a total of 18 fusion polypeptide chains); decamers (comprising 10 fusion proteins, a total of 20 fusion polypeptide chains); undecamers (comprising 11 fusion proteins, a total of 22 fusion polypeptide chains); and dodecamers (comprising 12 fusion proteins, a total of 24 fusion polypeptide chains).

The skilled reader will appreciate that such high levels of polymerisation, where fusion proteins are present as hexamers or larger polymeric structures, have not previously been obtainable in respect of fusion proteins based upon murine IgG heavy chain constant regions.

That said, the compositions of the invention may comprise still higher proportions of the fusion protein of the invention in the form of hexamers or larger polymeric structures. Merely by way of example, a composition of the invention may comprise at least at least 35%, at least at least 40%, at least at least 55%, at least at least 50%, at least at least 55%, or at least at least 60% of the fusion proteins in the composition present in the form of polymeric structures comprising at least 6 fusion proteins.

The composition of the invention may take the form of a pharmaceutical composition, in which case the carrier will be a pharmaceutically acceptable carrier, diluent or excipient. Such compositions may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The phrase pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Alternatively, the composition of the invention may be one that is used for research or analytical purposes, rather than therapeutic aims, in which case the desired carrier can be selected with reference to the purpose for which the fusion proteins are to be used, while also bearing in mind the need to retain the capacity for the fusion proteins of the invention to polymerise.

The invention will now be further described in the following Examples.

EXAMPLES

DNA constructs were generated as shown in Figure 1.

The inventors synthesised the mouse lgG1 sequence (SEQ ID NO: 2) into which they substituted by mutagenesis methionine 309 for cysteine to generate M84C (SEQ ID NO: 3). The inventors also added the 18 amino acid tailpiece from human IgM (SEQ ID NO: 5) or human IgA (SEQ ID NO: 4) to the C-terminus to create SEQ ID NO: 3.

Plasmids were stably or transiently transfected into either CHO-K1 or HEK cells. Cells secreting proteins were determined by ELISA and immunoblotting using appropriate antimouse Fc antibodies (Figure 2). Proteins were purified by protein G affinity chromatography and analysed by SDS-PAGE (Figure 2). From Figure 2, it can be seen that the addition of a human IgA-tailpiece allows for the formation of mouse lgG1 multimers/oligomers which run at a molecular weight commensurate with human lgG1 multimers in which the IgAtailpiece is inserted (>250 kDa).

The inventors confirmed the native molecular weights of the mouse multimers by sizeexclusion chromatography (Figure 3). From Fig. 3 it can be seen that mouse multimers run at approximately 300 and 600 kDa, molecular weights most like representing hexamers and higher order multimers, and as seen by immunoblotting in Figure 2.

The inventors then generated constructs by PCR into which variable numbers of mouse lgG1 hinges had been inserted upstream of the Fc and in which an antigen was inserted at the N-terminus (Figure 4)

SEQ ID NO: 1 - amino acid sequence of human hexa-Fc

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRWSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGKLVLGPPLYNVSLVMSDTAGTCY

SEQ ID NO: 2 - amino acid sequence of mouse lgG1

GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS

TFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD

FFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

PGK

SEQ ID NO: 3 - amino acid sequence of mouse hexa Fc with M84C (underlined)

GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS

TFRSVSELPICHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD

FFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

PGKAVLGPPLYNVSLVMSDTAGTCY

SEQ ID NO: 4 - amino acid sequence of human IgA tailpiece

PTHVNVSWMAEVDGTCY

SEQ ID NO: 5 - amino acid sequence of human IgM tailpiece

PPLYNVSLVMSDTAGTCY

SEQ ID NO: 6 - amino acid sequence of human IgA tailpiece with N459A (underlined)

PTHVAVSWMAEVDGTCY

SEQ ID NO: 7 - amino acid sequence of human IgM tailpiece with N563A (underlined

PPLYAVSLVMSDTAGTCY

SEQ ID NO: 8 - amino acid sequence of human IgA tailpiece with C471A (underlined)

PTHVNVSWMAEVDGTAY

SEQ ID NO: 9 - amino acid sequence of human IgM tailpiece with C575A (underlined)

PPYNVSLVMSDTAGTAY

SEQ ID NO: 10 - amino acid sequence of human IgA tailpiece with N459A + C471A (underlined)

PTHVAVSWMAEVDGTAY

SEQ ID NO: 11 - amino acid sequence of human IgM tailpiece with N563A + C575A (underlined)

PPLYAVSLVMSDTAGTAY

SEQ ID NO: 12 - amino acid sequence of mouse hinge

GCKPCICT

SEQ ID NO: 13 - amino acid sequence of mouse 5-hinge

GCKPCICTSRGCKPCICTVDGCKPCICTGCKPCICTRSGCKPCICT

SEQ ID NO: 14 - amino acid sequence of mouse hinge with cysteine to alanine substitution

GAKPAIAT

SEQ ID NO: 15 - amino acid sequence of mouse 5-hinge with cysteine to alanine substitution

GAKPAIATSRGAKPAIATVDGAKPAIATGAKPAIATRSGCKPCICT

SEQ ID NO: 16 - mouse hexa Fc with IgA tailpiece amino acid sequence

GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS

TFRSVSELPICHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD

FFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

PGKAVLGPTHVNVSWMAEVDGTCY

SEQ ID NO: 17 - mouse hexa Fc with IgA tailpiece DNA sequence

GGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATG

TGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCA

GTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGC

ACTTTCCGCTCAGTCAGTGAACTTCCCATCTGTCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGG

TCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACA

GGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGAC

TTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGC

CCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGG

AAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCT

CCTGGTAAAGCAGTCCTAGGACCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCT

SEQ ID NO: 18 - mouse hexa Fc with IgM tailpiece amino acid sequence

GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS

TFRSVSELPICHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD

FFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

PGKAVLGPPLYNVSLVMSDTAGTCY

SEQ ID NO: 19 - mouse hexa Fc with IgM tailpiece DNA sequence

GGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATG

TGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCA

GTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGC

ACTTTCCGCTCAGTCAGTGAACTTCCCATCTGTCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGG

TCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACA

GGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGAC

TTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGC

CCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGG

AAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCT

CCTGGTAAAGCAGTCCTAGGACCCCCCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCT

AC

SEQ ID NO: 20 - human hexa-Fc DNA sequence

GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC

CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA

AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG

GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCTGCCTCCAGGACTGGCTGAATGGCAAGG

AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA

GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC

TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG

CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAG

AGCCTCTCCCTGTCTCCGGGTAAATTAGTCCTAGGACCCCCCCTGTACAACGTGTCCCTGGTCATGTCCGACA

CAGCTGGCACCTGCTAC

SEQ

mouse lgG1 DNA

GGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATG

TGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCA

GTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGC

ACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGG

TCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACA

GGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGAC

TTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGC

CCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGG

AAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCT

CCTGGTAAATGA

SEQ ID NO: 22 - mouse hexa Fc with M84C (underlined) DNA

GGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATG

TGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCA

GTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGC

ACTTTCCGCTCAGTCAGTGAACTTCCCATCTGTCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGG

TCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACA

GGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGAC

TTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGC

CCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGG

AAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCT

CCTGGTAAAGCAGTCCTAGGACCCCCCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCT

ACT GA

SEQ ID NO: 23 - DNA sequence of human IgA tailpiece

CCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCTAC

SEQ ID NO: 24 - DNA sequence of human IgM tailpiece

CCCCCCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTAC

SEQ ID NO: 25 - DNA of human IgA tailpiece with N459A (underlined)

CCCACCCATGTCGCTGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCTAC

SEQ ID NO: 26 - DNA sequence of human IgM tailpiece with N563A (underlined)

CCCCCCCTGTACGCCGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTAC

SEQ ID NO: 27 - DNA sequence of human IgA tailpiece with C471A (underlined)

CCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCGCCTAC

SEQ ID NO: 28 - DNA sequence of human IgM tailpiece with C575A (underlined)

CCCCCCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCGCCTAC

SEQ ID NO: 29 - DNA sequence of human IgA tailpiece with N459A + C471A (underlined)

CCCACCCATGTCGCTGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCGCCTAC

SEQ ID NO: 30 - DNA sequence of human IgM tailpiece with N563A + C575A (underlined)

CCCCCCCTGTACGCCGTGTCCCTGGTCATGTCCGACACAGCTGGCACCGCCTAC

SEQ ID NO: 31 - DNA sequence of mouse hinge

GGTTGTAAGCCTTGCATATGTACA

SEQ ID NO: 32 - DNA sequence of mouse 5-hinge

GGTTGTAAGCCTTGCATATGTACATCTAGAGGTTGTAAGCCTTGCATATGTACAGTCGACGGTTGTAAGCCTT

GCATATGTACAGGTTGTAAGCCTTGCATATGTACAAGATCTGGTTGTAAGCCTTGCATATGTACA

SEQ ID NO: 33 - DNA sequence of mouse hinge with cysteine to alanine substitution

GGTGCTAAGCCTGCCATAGCTACA

SEQ ID NO: 34 - DNA sequence of mouse 5-hinge with cysteine to alanine substitution

GGTGCTAAGCCTGCCATAGCTACATCTAGAGGTGCTAAGCCTGCCATAGCTACAGTCGACGGTGCTAAGCCTG

CCATAGCTACAGGTGCTAAGCCTGCCATAGCTACAAGATCTGGTTGTAAGCCTTGCATATGTACA

SEQ ID NO: 35 - amino acid sequence of a hinge sequence with an additional glycosylation site (underlined)

GCKPCNCT

SEQ ID NO: 36 - amino acid sequence of a hinge sequence with additional glycosylation sites (underlined)

GCKPCNCTSRGCKPCNCTVDGCKPCNCTGCKPCNCTRSGCKPCNCT

SEQ ID NO: 37 - amino acid sequence of a heavy chain constant region with additional glycosylation sites (underlined)

VPEVSSVFIFPPKPKDNLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTNQTQPREE

QFNSTFRSVSELPICHQDWLNGKEFKCRVNSAAFPAPINKTISKTKGRPKAPQNYTIPPPK

EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTTPIMDTDGSYFVYSKLNVQKSN

WEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKA

SEQ ID NO: 38 - amino acid sequence of an IgM tailpiece with additional glycosylation sites (underlined)

PPLYNVSLNMSDTAGTCY

SEQ ID NO: 39 - glycan sequon

N-X-S/T

SEQ ID NO: 40 - human IgM amino acid sequence (UniProtKB P01871)

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL

RGGKYAATSQVLLPSKDVMQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR

DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS

TLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST

KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER

FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD

VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCWAHEAL

PNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

SEQ ID NO:41 - human IgA amino acid sequence (UniProtKB P01876)

ASPTSPKVFPLSLCSTQPDGNWIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDAS

GDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSP

SCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLC

GCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEEL

ALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRV

AAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSWMAEVDGTCY

Claims (41)

1. A fusion protein comprising two chimeric polypeptide chains, wherein each chimeric polypeptide chain comprises:

• two immunoglobulin G (IgG) heavy chain constant regions, each derived from a mouse IgG 1 heavy chain constant region, and • an immunoglobulin tailpiece from, or derived from, a non-native immunoglobulin, wherein the amino acid sequence of each of the IgG heavy chain constant regions comprises an amino acid modification which promotes the polymerisation of the fusion protein.

2. A fusion protein according to claim 1, wherein the amino acid modification of the IgG heavy chain constant region which promotes the polymerisation of the fusion protein comprises a modification of the amino acid residue corresponding to M84 of SEQ ID NO: 2.

3. A fusion protein according to claim 2, wherein the amino acid modification of the IgG heavy chain constant region which promotes the polymerisation of the fusion protein consists of a modification at an amino acid residue corresponding to residue 84 of the reference sequence of SEQ ID NO: 2.

4. A fusion protein according to any preceding claim, wherein the amino acid modification of the IgG heavy chain constant region which promotes the polymerisation of the fusion protein comprises substitution of an amino acid residue.

5. A fusion protein according to any preceding claim, wherein the substitution is by a cysteine residue.

6. A fusion protein according to claim 5, comprising a cysteine substitution of the residue corresponding to M84 of SEQ ID NO: 2.

7. A fusion protein according to any preceding claim, wherein the amino acid modification of the IgG heavy chain constant region which promotes the polymerisation of the fusion protein comprises deletion of an amino acid.

8. A fusion protein according to any preceding claim, wherein the amino acid modification of the IgG heavy chain constant region which promotes the polymerisation of the fusion protein comprises addition of an amino acid.

9. A fusion protein according to any preceding claim, wherein the immunoglobulin tailpiece is from, or derived from, an immunoglobulin selected from the group consisting of: IgA; and IgM.

10. A fusion protein according to any preceding claim, wherein the immunoglobulin tailpiece is a human tailpiece, or is derived from a human tailpiece.

11. A fusion protein according to any preceding claim, wherein the immunoglobulin tailpiece is modified as compared to the wild type tailpiece from which it is derived.

12. A fusion protein according to claim 11, wherein the modification of the immunoglobulin tailpiece is a modification that promotes polymerisation of the fusion protein.

13. A fusion protein according to any preceding claim, wherein the modification of the immunoglobulin tailpiece is a modification of an amino acid residue selected from the group consisting of: an amino acid residue corresponding to C17 of SEQ ID NO: 4; an amino acid residue corresponding to C17 of SEQ ID NO: 5; an amino acid residue corresponding to N5 of SEQ ID NO: 4, and an amino acid residue corresponding to N5 of SEQ ID NO: 5.

14. A fusion protein according to claim 13, wherein the modification of the amino acid residue corresponding to C17 of SEQ ID NO: 4 or of SEQ ID NO: 5 is a substitution or deletion of a cysteine residue.

15. A fusion protein according to claim 13, wherein the modification of the amino acid residue corresponding to N5 of SEQ ID NO: 4 or of SEQ ID NO: 5 is a substitution or deletion of an asparagine residue.

16. A fusion protein according to any preceding claim, wherein each chimeric polypeptide chain comprises a plurality of hinge regions.

17. A fusion protein according to any preceding claim, wherein each chimeric polypeptide chain comprises a number of hinge regions selected from the group consisting of: two hinge regions, three hinge regions, four hinge regions, five hinge regions, and six hinge regions.

18. A fusion protein according to claim 17, wherein one or more of the plurality of hinge regions is, or is derived from, a murine hinge region.

19. A fusion protein according to claim 18, wherein one of the plurality of murine hinge regions comprises the amino acid sequence GCKPCICT.

20. A fusion protein according to any of claims 1 to 18, wherein the murine hinge region comprises the amino acid sequence GAKPAIAT.

21. A fusion protein according to any preceding claim, wherein the fusion protein further comprises a targeting moiety.

22. A fusion protein according to claim 21, wherein the targeting moiety is selected from the group consisting of: an antigen, or antibody-binding portion thereof; an antibody, or antigen-binding portion thereof; an affimer; an aptamer; an adhiron; a DNA molecule; an RNA molecule; a protein; and a small molecule.

23. A fusion protein according to claim 22, wherein the antigen-binding portion of the antibody is an scFv sequence.

24. A fusion protein according to any preceding claim, wherein each chimeric polypeptide chain comprises a plurality of hinge sequences.

25. A fusion protein according to claim 24, wherein at least one of the plurality of hinge sequences lacks cysteine residues.

26. A fusion protein according to any preceding claim, wherein each polypeptide chain shares at least 80% identity with the amino acid sequence set out in SEQ ID NO: 16 or with the amino acid sequence set out in SEQ ID NO: 18.

27. A fusion protein according to any preceding claim, wherein each polypeptide chain comprises the amino acid sequence set out in SEQ ID NO: 16 or the amino acid sequence set out in SEQ ID NO: 18.

28. A fusion protein according to any preceding claim, wherein each polypeptide chain consists of the amino acid sequence set out in SEQ ID NO: 16 or the amino acid sequence set out in SEQ ID NO: 18.

29. A fusion protein according to claims 26 to 28, wherein the amino acid sequence of each polypeptide chain comprises one or more modifications as compared to the corresponding portion of the reference sequence (SEQ ID NO: 16 or SEQ ID NO: 18).

30. A fusion protein according to claim 29, wherein each polypeptide chain comprises no more than 10 amino acid modifications as compared to the corresponding portion of the reference sequence (SEQ ID NO: 16 or SEQ ID NO: 18).

31. A method of detecting the presence of a target molecule in a sample, the method comprising the steps of:

• contacting the sample with a fusion protein according to any preceding claim, • assaying binding of a target molecule to the fusion protein, and • thereby detecting the presence of a target molecule in the sample.

32. A method according to claim 31, wherein the method is selected from the group consisting of: a prognostic method, and a diagnostic method.

33. A method according to claims 31 or 32, wherein the method is carried out in vivo.

34. A method according to any of claims 31 to 33, wherein the sample is selected from the group consisting of: a tissue sample; and a body fluid sample.

35. A method according to any of claims 31 to 34, wherein the tissue sample is selected from the group consisting of: a muscle tissue sample; a skin tissue sample; a digestive tract tissue sample; a lung tissue sample; a breast tissue sample; a kidney tissue sample; a bone tissue sample; and a neural tissue sample.

36. A method according to any of claims 31 to 34, wherein the body fluid sample is selected from the group consisting of: a blood sample; a urine sample; a sputum sample; a bile sample; and a CSF sample.

37. A method according to any of claims 31 to 35, wherein the sample is a biopsy sample.

38. A method according to any of claims 31 to 37, wherein the sample is a human sample.

39. A method according to any of claims 31 to 38, wherein the target molecule is a biomarker.

40. A method according to any of claims 31 to 39, wherein the binding of the fusion protein of the invention to the target molecule is assayed by a technique selected from the group consisting of: immunofluorescence; enzyme-linked immunosorbent assay (ELISA); surface plasmon resonance (SPR) analysis; and immunoblotting.

41. A composition comprising fusion proteins according to any of claims 1 to 30, wherein at least 30% of the fusion proteins in the composition are present in the form of polymeric structures comprising at least 6 fusion proteins.

Intellectual Property Office

Application No: GB1804269.7