WO2008015480A2

WO2008015480A2 - Composition and method for modulating an immune response

Info

Publication number: WO2008015480A2
Application number: PCT/GB2007/050464
Authority: WO
Inventors: Camilo Colaco
Original assignee: Immunobiology Ltd
Current assignee: Immunobiology Ltd
Priority date: 2006-08-01
Filing date: 2007-08-01
Publication date: 2008-02-07
Anticipated expiration: 2009-02-01
Also published as: GB0615266D0; WO2008015480A3

Abstract

The present invention provides a polypeptide immunoconjugate comprising an antigenic polypeptide which is coupled to a polypeptide which is capable of binding to an Fc receptor. The antigenic polypeptide is derived from a pathogen or a pathogen product and as characterised in that it can be processed by the immune system in order to generate an immune response there against which confers protective immunity against the pathogen from which the antigenic polypeptide is derived. The invention further extends to methods for the treatment of a disease condition in a subject using the immunoconjugate of the invention.

Description

COMPOSITION AND METHOD FOR MODULATING AN IMMUNE

RESPONSE

Field of the Invention The present invention provides a novel immunoconjugate for use in mediating an immune response in a host. More specifically, there is provided a novel polypeptide immunoconjugate which can induce an immune response against an antigen in a host, said immune response being mediated through binding of the polypeptide immunoconjugate to Fc-receptor IV. More specifically, there is provided a fusion protein, which comprises a portion of the Fc domain of an immunoglobulin, for the treatment and/or prophylaxis of infection mediated by a pathogenic organism or a peptide or secreted product secreted there from.

Background of the Invention

The immune response is mediated by two complimentary mechanisms, the humoral response which involves soluble molecules, especially antibodies, and a cell mediated response which involves lymphocytes, particularly T-cells. It is now recognised that these two mechanisms also function together, with the utility of antibodies for antigen specific recognition and capture by receptors that bind the constant domains of antibodies, the Fc-Receptors (FcR). This has led to the proposed use of Fc fusion proteins which are designed to target Fc receptors in order to enhance immunogenicity. U.S. Patent No 7,067,110 discloses compositions which comprise pre-selected antigens which are fused to heavy chain constant regions of immunoglobulins.

However, there are a number of different Fc receptors, with each of these having specificity for a different heavy chain constant region of an immunoglobulin. Further, the different types of Fc receptor, when bound, can mediate different downstream signalling events, with these resulting in a variance in the mediated immune response. For example, some Fc receptors, when bound, have been shown to mediate a signal which activates an immune response, while other Fc receptors mediate a signal which inhibits the immune response (Ravetch J.V. and Clynes R.A. (1998) Divergent roles for Fc receptor and complement in-vivo. Annu Rev Immunol. 16. 421-432. Thus, while there are specific FcRs for the various antibody isotypes, the most important of these for activation of the immune response is thought to be the high affinity IgG receptor FcγRI or CD64 (Ravetch & Clynes 1998) and targeting of antigens to CD64 with antibodies against CD64 has demonstrated that these antigens are presented to the immune system in the context of both Class I and Class Il molecules (Ravetch & Clynes 1998). This has led to the use of targeting antigens for the production of vaccines for prophylactic and therapeutic use (e.g. U.S. Patent No 6,096,311 ). Moreover, this approach has been used for the development of both vaccines against infectious disease pathogens, as well as cancer (US Patent No 6,096,311 ) and other inflammatory conditions that require efficient antigen presentation (e.g. as described in WO99/41285).

An extension of this approach is the use of modified recombinant proteins containing antibody Fc domains to target antigens to CD64 (FcγRI). For example, WO02/058728 shows that antigens grafted onto antibodies containing the Fc heavy chain portion of human IgGI will stimulate cytotoxic T-cell responses to pathogens and tumour antigens. US Patent Application Publication Number 2005/0031628 shows that this method is not restricted to human Fc domains, but can also be mediated by Fc domains from other species that bind CD64. Nonetheless, much interest has focussed on the use of human IgGI Fc domains as these have been commonly used to provide fusion proteins of many proteins and so methods of their manufacture and purification are well known in the art.

However, a major limitation of the use of human Fc-IgGI fusion proteins is that they do not bind mouse CD64 and thus considerable effort has gone into the development of transgenic mice expressing the human form of CD64 to provide an animal model for the study of biologicals targeting CD64

The inventor of the present invention has now surprisingly identified that an immunoglobulin Fc protein-antigen fusion conjugate comprising a portion of the Fc domain derived from a human IgGI immunoglobulin attached to an antigen can mediate long term protective immunity in non- transgenic wild type mice against the pathogen from which the antigen is derived. The protective immune response is directed against the antigen component of the immunoconjugate which is typically a polypeptide fragment derived from the pathogenic organism, is surprisingly not mediated by means of binding to FcγRI (CD64), but rather is effected by binding to the murine FcγRIV receptor. That is, the binding of the immunoconjugate to the murine FcγRIV receptor results in antigen processing and down stream signalling which mediates an immune response in the host against the (antigenic) polypeptide fragment portion of the immunoconjugate.

Accordingly, following extensive experimentation, the present inventor has provided an immunoconjugate which can surprisingly induce an immune response which provides protective immunity, not only to a specific strain of a pathogen but to further strains of the same pathogen, which may result from events such as antigenic drift. Surprisingly, this immunity is substantially mediated through binding to FcγRIV and homologues thereof, binding through this receptor being necessary in order to mediate protective immunity in a host against the pathogen from which the antigen is derived.

The inventor has identified that the immunoconjugate of the invention has a particular utility in mediating protective immunity in a host against infectious diseases, such as influenza, where events such as antigenic shift and antigenic drift cause variance in the infectious agent which causes disease. Moreover, the immunoconjugate of the invention also has a specific utility in the induction of immune responses against pathogens that show high rates of sequence mutation and variation.

Summary of the Invention

According to a first aspect of the present invention there is provided a polypeptide immunoconjugate comprising at least one antigenic peptide or a fragment thereof complexed to an Fc receptor (FcR) binding peptide, said FcR binding peptide being capable of specifically binding to the FcγRIV receptor or a homologue thereof with an affinity sufficient to cause internalisation of the bound Fc Receptor.

As defined herein, a 'homologue' of the FcγRIV receptor is an FcR receptor which is encoded by a gene which is related to the gene which encodes the murine FcγRIV. Typically the FcγRIV homologue has a conserved function to that of FcγRIV. In particular, the homologue may be bound by the Fc portion of the constant domain of an antibody, or by a fragment of the Fc portion of the antibody.

The amino acid sequence for murine FcγRIV is provided in SEQ ID NO:1 :

MWQLLLPTAL VLTAFSGIQA GLQKAWNLD PKWVRVLEED SVTLRCQGTF SPEDNSIKWF HNESLIPHQD ANYVIQSARV KDSGMYRCQT ALSTISDPVQ LEVHMGWLLL QTTKWLFQEG

DPIHLRCHSW QNRPVRKVTY SQNGKGKKYF HENSELLIPK ATHNDSGSYF CRGLIGHNNK SSASFRISLG DPGSPSMFPP WHQITFCLLI GLLFAIDTVL YFSVRRGLQS PVADYEEPKI QWSKEPQDK

In certain embodiments the homologue of the murine Fc receptor FcγRIV is the human Fc receptor FcγRllla (CD16a).

The amino acid sequence for the human homologue of FcγRIV, FcγRllla (CD16a) is provided below as SEQ ID NO:2:

MWQLLLPTAL LLLVSAGMRT EDLPKAWFL EPQWYRVLEK DSVTLKCQGA YSPEDNSTQW

FHNESLISSQ ASSYFIDAAT VDDSGEYRCQ TNLSTLSDPV QLEVHIGWLL LQAPRWVFKE

EDPIHLRCHS WKNTALHKVT YLQNGKGRKY FHHNSDFYIP KATLKDSGSY FCRGLVGSKN

VSSETVNITI TQGLSVSTIS SFFPPGYQVS FCLVMVLLFA VDTGLYFSVK TNIRSSTRDW KDHKFKWRKD PQDK

In certain embodiments, the homologue of FcγRIV is an orthologue of the FcγRIV receptor, that is that the genes which encode for the Fc receptors (FcRs) are genes in different species which have evolved from a common ancestral gene by speciation. Typically the orthologue retains the same function in the course of evolution.

As such, in certain embodiments, the homologue of FcγRIV is an orthologue, said orthologue typically being FcγRllla (CD16a).

The term "specifically binds" or "binding specificity" refers to the ability of the FcR binding peptide to bind to a target epitope present on the FcγRIV receptor or the homologue thereof with a greater affinity than it binds to a non-target epitope. In certain embodiments, specific binding refers to binding of the FcR binding peptide to a target epitope present on the FcγRIV receptor with an affinity which is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target epitope. In certain embodiments, binding affinity may be determined by a number of means, such as, but not limited to an affinity ELISA assay, a BIAcore assay or by a kinetic method. An "epitope" refers to a portion of the FcγRIV receptor which is capable of being recognised by, and bound by, a binding compound such as an FcR binding peptide.

Typically, binding of the FcγRIV receptor by the FcR binding peptide mediates signalling from the FcγRIV receptor which results in an upregulation of the immune response. In humans, examples of homologues of FcγRIV are encoded for by the genes which encode for the Fc receptor, FcγRllla (CD16). Without wishing to be bound by theory, the inventor predicts that as yet unidentified homologous receptors and orthologues may be found which are encoded by genes located on human chromosome 1q21 to chromosome 1q23 and further from synthetic regions thereof, it is predicted that such homologous receptors will have a charged amino acid residue in the transmembrane region. It is further predicted that such homologous receptors will exhibit homology at a site equivalent to the N-glycosylated Asparagine residue present at position 162 of the FcγRIV receptor.

In certain embodiments, the FcR binding peptide comprises an amino acid sequence relating to the CH2 (C2) constant domain derived from the Fc portion of the heavy chain of a human immunoglobulin. In certain embodiments the FcR binding peptide comprises an amino acid sequence derived from the CH2 (C2) constant domain of the Fc portion of the heavy chain of a human immunoglobulin.

In certain embodiments the FcR binding peptide comprises an amino acid sequence derived from the CH3 (C3) constant domain of the Fc portion of the heavy chain of a human immunoglobulin. In certain embodiments the FcR binding peptide comprises an amino acid sequence derived from the CH2 (C2) constant domain along with an amino acid sequence derived from the CH3 (C3) constant domain of the Fc portion of the heavy chain of a human immunoglobulin.

In certain embodiments, the CH2 (C2) and / or CH£ (C3) constant domains of the FcR binding peptide of the immunoconjugate are derived from the heavy chain of a human immunoglobulin, in particular IgG, most preferably IgGI or lgG3.

In certain further embodiments, the FcR binding peptide is derived from any immunoglobulin which has a heavy chain which has binding specificity to the FcγRIV or a homologue thereof. In certain embodiments the FcR binding peptide is derived from the heavy chain of a human immunoglobulin. In certain embodiments, the immunoglobulin is of the subclass IgG. In where the Fc receptor binding protein is derived from the heavy chain of human IgG, typically the IgG is of the subclass IgGI or of the subclass lgG3.

In certain further embodiments, the Fc receptor binding peptide comprises the CH2 constant domain of the IgGI human immunoglobulin or the CH2 constant domain of the lgG3 human immunoglobulin. The CH2 constant domain of human IgGI or human lgG3 is also known as the Cγ2 domain or the C2 domain, this alternative nomenclature being derived from the heavy chain of human IgG being designated the Y chain. In certain embodiments, the whole, or a part of the CH3 (C3) domain is provided along with the CH2 domain. As defined herein, the term 'internalisation' means endocytosis and, in particular, receptor mediated endocytosis or Fc mediated phagocytosis. Endocytosis is the process of the uptake of macromolecules or particles into cells. In relation to the present invention, internalisation particularly occurs when an Fc receptor is bound by a ligand, for example an Fc receptor binding peptide, with a sufficiently high binding affinity that internalisation of the Fc receptor and the bound ligand occurs.

By the term 'sufficiently high binding affinity' it is meant that the Fc Receptor (FcR) is bound by its ligand with a binding affinity which causes the internalisation of the FcR from the cell membrane and its translocation within the cell. This has the result that the immunoconjugate which is bound to the FcR is internalised within the cell and is processed by the cell's antigen processing pathway. This processing results in the display of the antigenic peptide of the immunoconjugate or of fragments thereof which have been broken down during antigen processing and which are displayed to the cells of the immune system by major histocompatability molecules (MHC).

In certain embodiments, in order to induce internalisation, typically the Fc receptor binding peptide component of the immunoconjugate binds to the Fc receptor with a dissociation rate constant binding affinity of from about 10^"6Kd to about 10^"9Kd. In certain embodiments, the binding affinity between the Fc receptor binding polypeptide and the Fc receptor is from about 10^"8Kd to about 10^"9Kd. In certain embodiments, the association rate constant between the Fc receptor binding polypeptide of the fusion protein of the invention and the Fc receptor will be in the region of from about IxIO⁶Ka to about 3x10⁹Ka. The antigenic peptide (which may also be referred to as an antigenic polypeptide) may be conjoined to the FcR binding peptide (which may also be referred to as the FcyR (Fc gamma receptor) binding peptide) at either its N- (amino) or C- (carboxyl) terminal. In certain embodiments, the amino terminal region of the immunoconjugate defines the amino acid sequence encoding for the antigenic peptide, while the carboxyl terminal region comprises an amino acid sequence encoding for the FcR binding portion.

In certain embodiments, a linker moiety or amino acid spacer may be used to conjoin the FcR binding peptide with the antigenic peptide of the immunoconjugate at any suitable amino acid residue that does not affect its FcR binding capacity. The linker functions to allow the individual peptide component of the immunoconjugate (i.e the antigenic peptide, which is at least one antigenic peptide fragment, and the FcR binding peptide) to fold independently when part of the fusion protein immunoconjugate, thus allowing them to retain their tertiary structure and hence associated specificity and activity. It is of particular importance that the tertiary structure of the FcR binding portion of the immunoconjugate is not substantially altered when the FcR binding peptide is provided as part of the immunoconjugate, as a change in tertiary structure may result in an impairment of FcR binding properties. Preferred forms of the linker moiety, which may for example be a hinge, are described hereinafter.

The polypeptide immunoconjugate of the invention may be defined by the primary amino acid sequence ABC, wherein A is a polypeptide having the amino acid sequence which defines at least one antigenic peptide or a fragment thereof, B is an optional linker moiety and C is an amino acid sequence defining at least one peptide or fragment thereof which has an FcR binding domain and which binds to FcγRIV or homologues thereof with a binding affinity sufficient to cause internalisation of the FcR and its bound ligand.

In certain embodiments, A defines the N terminal (amino) portion of the peptide and C defines the C-terminal (carboxyl) terminal of the polypeptide. In certain embodiments, the antigenic peptide A is derived from a pathogenic organism. In certain embodiments, the pathogenic organism is a bacterial pathogen. In certain embodiments, the pathogenic organism is a viral pathogen.

In certain embodiments where the pathogenic organism is a bacterial pathogen, said bacterial pathogen may be selected from the group comprising, but not limited to; tuberculosis, typhoid, anthrax, bacterial meningitis, cholera, diphtheria, gonorrhoea, legionellosis, leptispirosis, listeriosis, MRSA infection or pertussis.

In certain embodiments where the pathogenic organism is a viral pathogen, said viral pathogen may be selected from the group comprising, but not limited to; influenza, rhinovirus, coronavirus, HIV, human papillomavirus, smallpox, rabies, rubella, hepatitis, herpes simplex virus, herpes zoster virus, viral meningitis, yellow fever, west Nile disease, chicken pox (varicella), dengue, SARS or foot and mouth disease virus (FMDV).

In certain embodiments, where the antigenic peptide is derived from a viral pathogen, the viral pathogen is influenza and the antigenic peptide is derived from haemagglutinin (H, HA) or neuraminidase (NA, N). In certain embodiments, the antigenic peptide is derived from the haemagglutinin of influenza, wherein the haemagglutinin is HA1 , HA2, HA3, HA5, HA7 or HA9. In certain embodiments where the antigenic peptide is derived from haemagglutinin, the amino acid sequence may relate to a haemagglutinin- derived protein which has been genetically modified due to an event such as antigenic drift wherein a change in the amino acid sequence of the haemagglutinin protein results.

In certain embodiments, the antigenic peptide comprises a peptide derived from aino acid residues 17 to 530 of HA3, HA5 or further from HA1 , HA2, HA7 or HA9.

In certain embodiments, the antigenic polypeptide may be derived from a secreted product of a bacterial pathogen. Examples of such excreted products include those selected from the group comprising; leukocidins, streptolysin S, streptolysin O, NADase, hyaluronidase, streptokinases, and pyrogenic exotoxins.

In certain embodiments, the antigenic polypeptide A is a peptide fragment of a protein derived from a pathogen which is causative of a pathogenic process, said pathogenic process causing illness or disease in a host. In certain embodiments, the antigenic polypeptide A is an infectious disease target.

In certain embodiments, the antigenic polypeptide A is derived from a secreted product or other infectious agent which is derived from a pathogenic organism and causative of illness or disease.

In certain embodiments the antigenic fragment A is derived from a protein involved in a disease process, wherein presentation of the antigenic peptide or a fragment thereof to the cells of the immune system is sufficient to mediate an immune response in the host sufficient to prevent or treat said disease process.

In certain embodiments, the antigenic polypeptide A is derived from a protein that is responsible for, or which contributes to, a non-pathogenic inflammatory condition including, but not limited to: arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), atherosclerosis, stroke, psoriasis, cardiac inflammation, allograft rejection, acute and chronic graft versus host disease, and tissue damage resulting from insult or injury. In certain embodiments, the antigenic polypeptide may be derived from the agents which are causative of, or result from the onset of; Alzheimer's disease (AD) where the antigen may be beta amyloid peptide(s) or amyloid precursor protein (APP), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

In certain embodiments, the immunoconjugate may further comprise, or be provided in a composition along with an adjuvant and/or an immunomodulator. In certain embodiments, the immunomodulator may be a proinflammatory cytokine which serves to modulate the immune response.

Suitable adjuvants and immunomodulators are readily apparent to the person skilled in the art, such as Freund's complete or incomplete adjuvant, alum, MF59, QUIL A™ (Quil A Saponin), Detox, ISCOMs, cytokines or squalene. In certain embodiments, the Fc Receptor (FcR) binding polypeptide (C) may be a series of amino acid residues which are derived from at least one constant domain of the Fc portion of the heavy chain of an immunoglobulin molecule. In certain embodiments, the immunoglobulin is derived from the same species that is to be treated with the immunoconjugate in order to minimise the occurrence of the Fc receptor binding peptide portion of the immunoconjugate being immunogenic. Thus, for treating human diseases, an Fc domain or an amino acid sequence derived from a human immunoglobulin would be preferred.

Different classes of human immunoglobulin may be suitable for use in the present invention. The class or 'isotype' of an antibody is defined by its heavy chain, and in particular the sequence of the heavy chain region. In particular, immunoglobulins of the isotype IgG are most preferred, however antibodies of isotypes IgA, IgM, IgE and IgD may also have utility in various further embodiments of the present invention.

In certain embodiments, the antibody is of the isotype IgG. IgG has a number of subclasses, such as IgGI , lgG2a, lgG2b, and lgG3. Each IgG subclass has a very high level of homology in the constant domains, but each differs significantly in the hinge region. Although any subclass of IgG has utility in the present invention, in certain embodiments of the present invention the subclasses are IgGI or lgG3.

The inventor has identified that the structure of the Fc portion of the human lgG3 antibody is particularly suited to the present invention, due to the presence of the extended hinge region. The hinge region of an antibody is generally located between the CH1 (C1 ) constant domain and the CH2 (C2) constant domain and is though to provide structural flexibility to the antibody molecule to facilitate binding by the Fab portion of the antibody.

The constant domains of an antibody are known to have importance in directing the immune response, and in particular the recruitment of effector functions which mediate the immune response following antibody binding. The type of effector functions which are induced following the binding of an antibody can be dependent upon the constant regions of the heavy chain and in particular the CH2 and CH3 domain regions. The ability to induce a response from the immune system which provides long term protective immunity is important in the continued protection of a subject against a pathogen.

An integral part of such a response is the binding and activation of FcRs. FcRs are present on many cells of the immune system such as antigen presenting cells, and in particular macrophages, B cells, neutrophils, mast cells, NK cells and follicular dendritic cells. This binding results in the activation of a number of effector mechanisms such as the release of immune mediators such as cytokines, complement activation and ADCC (antibody dependent cell mediated cytotoxicity). FcRs bind with the

Fragment Constant (Fc) region of immunoglobulins. They are antibody class specific and isotype selective.

According to a further aspect of the present invention there is provided a method for mediating an immune response against an antigenic peptide in a mammalian host, the method comprising the steps of:

- providing a polypeptide immunoconjugate comprising at least one antigenic peptide or fragment thereof complexed to an FcR binding peptide which is capable of binding to the FcγRIV receptor or a homologue thereof with an affinity sufficient to cause internalisation of the FcγRIV receptor, and

- administering a therapeutically effective amount of said composition to a subject in need of such treatment.

In certain embodiments, the immunoconjugate is administered intravenously, subcutaneously or intramuscularly, these routes being preferred as these tissues contain dendritic cells which express Fc receptors and further said tissues lack high levels of serum IgG which may compete for Fc receptor binding.

In certain embodiments, the subject is a mammal, typically a human.

According to a further aspect of the present invention there is provided a method for inducing an immune response in a subject, the method comprising the steps of:

- providing an immunoconjugate comprising at least one antigenic polypeptide sequences and an Fc receptor binding polypeptide which binds to the murine FcγRIV receptor or a homologue thereof with a binding affinity sufficient to cause internalisation of the bound Fc receptor, and

- administering a therapeutically effective amount of said immunoconjugate to a subject in which the induction of the immune response against said antigenic polypeptide or a fragment thereof is desired.

In certain embodiments the method extends to inducing an immune response for the treatment of infection with a pathogenic disease, the method further comprising the steps of: - obtaining an antigenic peptide sequence derived from the pathogen which is causative of the disease or a product derived from the pathogen,

- using said antigenic polypeptide sequence in the formation of the immunoconjugate of the first aspect of the invention, and

- administering a therapeutically effective or prophylactically effective amount of a composition comprising the immunoconjugate to a subject in need of such treatment.

A further aspect of the present invention provides for the use of the polypeptide immunoconjugate of the present invention in the treatment and / or prophylaxis of an infectious disease in a subject.

In certain embodiments, the subject is a mammal, typically a human.

A yet further aspect of the present invention provides for the use of the polypeptide immunoconjugate of the present invention in the preparation of a medicament for the treatment and / or prophylaxis of an infectious disease in a subject.

A further aspect of the present invention provides for the use of the polypeptide immunoconjugate of the present invention in the treatment and / or prophylaxis of and influenza virus infection. A further aspect of the present invention provides for the use of the polypeptide immunoconjugate of the present invention in the treatment and / or prophylaxis of a hepatitis virus infection.

In yet a further aspect the present invention provides for the use of the polypeptide immunoconjugate of the present invention in the treatment and / or prophylaxis of a HIV infection.

According to a yet further aspect of the present invention there is provided the use of the polypeptide immunoconjugate of the present invention in the preparation of a medicament for the treatment or prevention of infection of a subject having an infectious disease.

In certain embodiments the infectious disease is type A influenza. In certain embodiments, the infectious disease is hepatitis, in particular type C hepatitis. In certain embodiments, the infectious disease is hepatitis, in particular, type B hepatitis. In certain embodiments, the infectious disease is AIDS, in particular that caused by the HIV virus.

The present inventors have also identified that the polypeptide immunoconjugate of the present invention may be administered along with a second anti-microbial composition for the treatment of infectious diseases. This provides a combination therapy which has utility in relation to a viral infection which has a particularly high pathogenicity.

Accordingly, a further aspect of the present invention provides a method for preventing or treating a microbial infection, the method comprising the steps of;

- providing a polypeptide immunoconjugate comprising an antigenic peptide or fragment thereof complexed to an FcR binding peptide capable of binding the FcγRIV receptor with an affinity sufficient to cause internalisation of the FcR to which it binds, - administering a therapeutically effective amount of said immunoconjugate to a subject in need of such treatment, and -further administering a therapeutically effective amount of a suitable secondary anti-microbial compound to the subject.

In one embodiment the antigenic fragment is derived from a pathogenic organism, wherein presentation of the antigenic peptide or a fragment thereof to the cells of the immune system is sufficient to mediate an immune response in the host sufficient to provide protective immunity to the host against the pathogen from which the antigenic fragment is derived.

In one embodiment a linker moiety or spacer may be provided between the antigenic peptide and the FcR binding peptide elements of the immunoconjugate. In a further embodiment, the immunoconjugate comprises a plurality of antigenic peptide fragments.

In one embodiment, the secondary anti-microbial compound is administered along with the immunoconjugate, however, in further embodiments, the secondary anti-microbial compound may be administered before or after the immunoconjugate has been administered.

In one embodiment, the pathogen is a viral pathogen and the secondary compound is an anti-viral. The secondary anti-viral compound may be selected from the group comprising; ribavirin, amantadine, rimantadine, oseltamivir (TAMIFLU™), zanamivir or cytokines including the interleukins. In one embodiment, the polypeptide conjugate antigen comprises an antigenic peptide derived from an influenza virus. In another embodiment, the antigenic peptide is derived from a hepatitis virus. In a further embodiments, the antigenic peptide is derived from HIV.

According to a yet further aspect of the present invention there is provided the use of the polypeptide immunoconjugate of the present invention and an anti-viral compound in the preparation of a combined medicament for the treatment or prevention of the infection of a subject with a viral pathogen.

In certain embodiments, the polypeptide conjugate of the invention may be administered as a medicament within an interruptive anti-viral therapy schedule in combination with anti-viral compounds.

In certain embodiments, the polypeptide conjugate of the invention may be administered as a medicament in interruptive anti-viral therapy of AIDS in combination with triple-therapy anti-viral compounds.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context clearly demands otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well as two or more different active agents in combination, while references to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

The nomenclature used to describe the polypeptide constituents of the fusion protein of the present invention follows the conventional practice wherein the amino group (N) is presented to the left and the carboxy group to the right of each amino acid residue.

The expression "amino acid" as used herein is intended to include both natural and synthetic amino acids, and both D and L amino acids. A synthetic amino acid also encompasses chemically modified amino acids, including, but not limited to salts, and amino acid derivatives such as amides. Amino acids present within the polypeptides of the present invention can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the circulating half life without adversely affecting their biological activity.

The terms "peptide", "polypeptide" and "protein" are used herein interchangeably to describe a series of at least two amino acids covalently linked by peptide bonds or modified peptide bonds such as isosteres. No limitation is placed on the maximum number of amino acids which may comprise a peptide or protein. Furthermore, the term polypeptide extends to fragments, analogues and derivatives of a peptide, wherein said fragment, analogue or derivative retains the same biological functional activity as the peptide from which the fragment, derivative or analogue is derived Furthermore the term "fusion protein" as used herein can also be taken to mean a fusion polypeptide, fusion peptide or the like, or may also be referred to as an immunoconjugate. The term "fusion protein" refers to a molecule in which two or more subunit molecules, typically polypeptides, are covalently or non-covalently linked.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

Brief description of the figures The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention wherein:

Figure 1 shows a chromosome map of a number of species showing the location of genes encoding Fc Receptors,

Figure 2 shows the alignment of the amino acid sequences of murine FcγRIV with human FcγRllla. The charged residue Q (emboldened and underlined) forms a salt bridge with the gamma chain which is crucial for the functioning and internalisation of the Fc Receptor,

Figure 3 shows a graph showing weight change of mice post- infection with heterologous virus, Figure 4 shows virus titres in nasal washes of mice vaccinated with a pandemic HA(H5)-Fc immunoconjugate,

Figure 5 shows the binding of HA-Fc immunoconjugate (graph A) and gp120-Fc immunoconjugate (graph B) to human THP1 cells. Human IgGI was used as a control,

Figure 6 shows binding of HA-Fc immunoconjugate (graph A) and gp120-Fc immunoconjugate (graph B) to murine RAW cells.

Human IgGI was used as a control,

Figure 7 shows the results of experimentation assessing the binding of immunoconjugates to mannose receptor,

Figure 8 shows the immunogenicity (humoral immunity) of immunoconjugates assessed by antibody production (chart A) and haemagglutination inhibition (chart B). Group 1 is a negative control (IgGI ), Group 2 is a positive control (live virus), Group 3 is the HA-FcLL immunoconjugate, and Group 4 is the HA-FcVA immunoconjugate, and

Figure 9 shows survival of HA(H5)-Fc immunized mice and non- immunised control mice (negative control) following challenge with a pandemic avian influenza strain.

Detailed Description of the Invention

An Fc receptor is a protein which is found on the surface of certain cells which have involvement in the functioning of the immune system, such as macrophages, natural killer (NK) cells, neutrophils and mast cells. The term "Fc" relates to the function of the Fc receptor binding ligand, which is the part of an antibody known as the Fc (fragment, crystallization) portion. There are a number of types of Fc receptor. Each Fc receptor type is classified with regard to the type of antibody which is recognised by it. Those that bind the most common class of antibody, that is antibodies of the IgG subclass, are called Fc gamma receptors (FcyR). FcyRs belong to the immunoglobulin superfamily. This class of Fc receptor are involved with pahgocytosis of opsonised antigens, such as microbes.

The human family of FcyR receptors includes: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRllla (CD16a), and FcγRlllb (CD16b).

The utility of the present invention lies in the ability of the immunoconjugate of the present invention to bind to FcγRIV receptors with a sufficiently high affinity so as to induce the internalisation of the FcR within the cell upon which the FcR is present. This binding, results in the activation of a number of effector mechanisms such as the release of immune mediators, such as chemokines and cytokines. Further, the binding of the FcR by the constant domain fragment causes internalisation and activation of the FcR.

Following FcR internalisation, the bound immunoconjugate is also internalised and is broken down and processed in the cell's antigen presentation pathway, this resulting in the presentation of the conjoined antigenic polypeptide and fragments thereof.

Previous patents have described the binding to FcγRI as a means of enhancing immunogenicity of antigens by the use of bifunctional agents containing antibodies against FcRs (Medarex, WO 96/40788). These reagents bind to the FcR but do not induce their internalisation for antigen presentation (T.Keler et.al. (2002) J.Immunol. 165: 6738-6742,). The internalisation of FcRI is induced by the binding of immune complexes and is mediated by the binding of immunoglobulin to the FcR via the Fc domain (A.Yada et al. (2003) Cell lmmunol.225 (1 ) : p21-32, PT. Harrison et al. (1994) J. Biol. Chem. 269 (39): 24396- 24402). It has been further shown that this can be mimicked by the use of modified intact immunoglobulin containing foreign sequences introduced into the variable domains by CDR-grafting (WO 02/058728). The present inventor has identified immunocongugates which bind to FcγRIV and which cause the internalisation of the FcR. This internalisation results in the uptake and presentation of the antigenic peptide which is provided as part of the immunoconjugate, this allowing said antigenic peptide or fragments thereof, to be displayed to the cells of the immune system.

There are four known types of FcR known to be specific to IgG, with each have a specific function ascribed to them. FcγRI (also known as CD64) is known to be present on macrophages and monocytes. FcγRII (CD32) is known to be expressed on B cells, macrophages, neutrophils, and monocytes. FcγRIII (CD16) is known to be expressed by macrophages, NK cells and neutrophils. Lastly FcγRIV is present on cells of a monocytic lineage. The human homologue of FcγRIV is thought to be FcγRllla (CD16a) and the present invention includes this receptor and other human homologues (Nimmerjahn et.al. 2005). This includes homologues located between FcγRIIA and FcγRIIB on human chromosome 1 as well as other homologous FcR genes that contain transmembrane sequences containing a charged residue capable of salt bridging to the FcR γ-chain which mediates internalisation of the FcR (Nimmerjahn and Ravetch 2006). FcRs specific to other antibody isotypes include; FcεRI and FcεRII which are expressed by B cells, monocytes and follicular dendritic cells and which have specificity for IgE Fc portions; FcαR which has specificity for IgA Fc portions and FcμR which has specificity for IgM Fc domains.

It is known that different areas of the Fc portion of the constant domain of the heavy chain of an immunoglobulin bind to different and distinct regions of the FcR. Mutations of various residues have been shown to affect the affinity of binding between the Fc portion of an immunoglobulin and an appropriate FcR (Canfield and Morrison, J. Exp Med, VoI 173, June 1991 , p1483). Moreover, it is thought that in immunoglobulins, the N- glycosylated asparagine residue at position 297 interacts with a carbohydrate residue at position 162 of the FcγRllla and the crystal structure of immunoglobulin / FcR binding elucidated by Sonderman et al. (Sonderman P and Oosthuizen V, Biochemical Society Transactions (2002), VoI 30, part 4) shows that the carbohydrate residue of the immunoglobulin interacts with the FcR during binding. It is thus expected that the preferred fusion proteins of the present invention would be made in a mammalian system in order to ensure correct glycosylation. Nonetheless, it has surprisingly been observed by the present inventor that the immunoconjugates of the present invention, when made in non- mammalian systems, such as insect or yeast cells, result in more biologically active species. Accordingly, the inventor predicts that the presence of an incompletely processed N-glycan at position 297 or replacement with an O-glycan can increase the affinity of binding to the FcR. The inventor further predicts that other mutations in this region such as changing the flanking serine or tyrosine residues would also increase the affinity of FcR binding.

In one embodiment, the modification in the region of position 297 relates to the substitution of the serine residue equivalent to position 298 to an alanine residue or a glycine residue. This substitution serves to introduce an amino acid residue which is less bulky than the serine residue and accordingly serves to reduce the steric hinderance during the binding of the FcR binding peptide portion of the immunoconjugate with the FcR.

Accordingly in certain embodiments, the FcR binding peptide portion of the immunoconjugate binds to the FcγRIV receptor or a homologue thereof, with a binding affinity sufficient to cause internalisation of the Fc receptor. In further embodiments, the FcR binding protein binds to at least one of the FcRs selected from the group comprising; FcγRI (CD64), FcγRII (CD32), or FcγRIII (CD16) with a binding affinity sufficient to cause internalisation of the FcR. In certain embodiments, the FcR binding portion can bind to both murine FcγRIV and human FcγRllla (CD16a). In a yet further embodiment, the Fc domain binds to a receptor which is a homologue of FcγRIV.

The amino acid sequence for murine FcγRIV is provided in SEQ ID NO:1. The amino acid sequence for the human homologue of FcγRIV, FcγRllla (CD16a) is provided as SEQ ID NO:2.

Figure 1 shows a map of chromosome 1 of a number of species showing the location of the genes encoding FcR's. Figure 2 shows the result of an alignment of the amino acid sequences of mouse FcγRIV with the human FcγRllla.

As discussed above, the present invention extends to the use of receptors which are homologues of mouse FcγRIV with the human FcγRllla. Such homologous receptors may be found on chromosome 1q21 to chromosome 1q23. Further, such homologous receptors would likely have a charged amino acid residue in the transmembrane region. It is further predicted that such homologous receptors will exhibit homology at a site equivalent to the N-glycosylated asparagine residue at position 162 of the FcγRIV receptor.

Figure 2, which shows an alignment of mouse FcγRIV and human FcγRllla, shows the charged residue (Q) present in the transmembrane region (underlined).

In certain embodiments, the invention further extends to functionally similar receptor homologues of FcγRIV which have at least 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence homology to the amino acid sequence of SEQ ID NO:1.

In certain embodiments, the invention further extends to functionally similar orthologues of FcγRIV which have at least 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence homology to the amino acid sequence of SEQ ID NO:1.

In certain embodiments, the FcR binding peptide portion of the immunoconjugate of the present invention is comprised of the CH2 constant domain, including the hinge region, of the IgGI antibody or a fragment thereof. The sequences which encode the CH2 domain can be found in the Kabat database of sequences (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed., United States, Public Health Service, National Institutes of Health, Bethesda). In certain embodiments, the CH2 constant domain (also referred to as the C2 constant domain), is derived from the heavy chain of the human IgGI antibody. In certain embodiments, the CH2 domain of the immunoglobulin is modified in the region of the N-glycosylation site at position 297. In one embodiment the Fc receptor binding protein comprises the amino acid sequence of SEQ ID NO:3. As such, the fusion protein of said embodiment comprises one or more antigenic polypeptide sequences along with an Fc receptor binding polypeptide comprising the amino acid sequence of SEQ ID NO:3.

In a still further embodiment, the Fc receptor binding protein comprises a fragment, variant or derivative of the sequence as shown in SEQ ID NO:3, wherein said fragment, variant or derivative has the biological activity of the polypeptide having SEQ ID NO:3.

SEQ ID NO:3:

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKATPPVLDSDGSFFLYSKLTVDKGRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

In particular, SEQ ID NO:1 details a polypeptide which can bind to an Fc receptor. The sequence can be aligned with residues 216 to 447 of the determined sequence for the heavy chain constant region of IgG as defined on the Kabat Database of Sequences of Proteins of Immunological Interest (www.kabatdatabase.com). On the basis that the first amino acid reside of SEQ ID NO:3 is taken to be numbered as residue 216 and the last residue numbered as residue 447, then an asparagine (Asn, N) residue, which can be glycosylated, is provided at residue 297. The glycine residue (G, GIy) at residue 415 of SEQ ID NO:3 represents a substitution of the serine (S, Ser) residue present in the defined Kabat database sequence. SEQ ID NO:3 further comprises an alanine (A, Ala) residue at position 393 which replaces the threonine residue (Thr, T) provided in the defined Kabat database sequence. The sequence of SEQ ID NO:3 may be further mutated such that the serine (Ser, S) residue at position 298 can be substituted with an alanine residue (Ala, A). Furthermore, in order to create an mutant version of the fusion protein which comprises the sequence of SEQ ID NO:3 as the Fc receptor binding polypeptide, the two leucine residues provided at residues 234 and 235 of SEQ ID NO:3 may be mutated, for example to residues such as valine and alanine respectively, in order to impair Fc receptor binding.

In certain embodiments, the FcR binding portion of the immunoconjugate is comprised of the CH2 constant domain of the lgG3 antibody or a fragment thereof. In certain embodiments, the CH2 constant domain (also referred to as the C2 constant domain), is derived from the Fc portion of the heavy chain of the human lgG3 antibody. In certain embodiments the CH2 domain of the lgG3 is modified in the region of the N-glycosylation site at position 297.

In certain embodiments, the FcR binding peptide comprises the CH3 (C3) constant domain of an immunoglobulin, in particular an antibody of the subtype IgG, and in particular of the subclass IgGI or lgG3.

The immunoconjugate may be formed by the conjugation of the antigenic peptide portion conjoined to the FcR binding portion by any suitable chemical or molecular genetic technique.

The immunoconjugate can therefore be defined as a fusion protein. A fusion protein can be created through genetic engineering, this resulting in the joining of 2 or more peptides. The fusion protein is formed by creating a fusion gene by removing the stop codon from the DNA sequence of first protein and then appending the DNA sequence encoding for the second protein in frame with the reading sequence of the codons of the first DNA sequence. The DNA sequence can be transfected into a host cell and expressed as a fusion protein for use or used directly as a DNA construct.

In certain embodiments, a linker moiety may be used between the first and second peptide. This linker may be, for example, a hinge region. The hinge region serves not only to link the antigenic peptide with the FcR binding component, but also provides increased flexibility of the immunoconjugate which can confer improved binding specificity, particularly as the linker can space the 2 components of the fusion protein so as to allow them to assume their normal tertiary structure.

In another embodiment the antigenic peptide may be linked to the linker moiety at either its N-(amino) or C-(carboxyl) terminal or at any suitable amino acid residue that does not affect the FcR binding capacity of the FcR binding peptide domain of the immunoconjugate.

In certain embodiments, the FcR binding peptide domain of the immunoconjugate may be obtained by recombinant methods. In certain embodiments, the FcR binding domain may be obtained following proteolytic digestion of immunoglobulin molecules, for example by papain digestion of immunoglobulins.

In certain embodiments, the FcR binding peptide is conjugated to the antigenic peptide by chemical methods. Such conjugation and linkage techniques would be well known to those skilled in the art and may include, for example, conjugation by thio-ester crosslinking utilising cysteine residues of the Fc polypeptide. In a further embodiment utilising cysteine residues of the FcR polypeptide, the antigenic fragment may be linked by to the FcR binding peptide via cysteine residues present in the antigenic peptide. In a yet further embodiment the conjugation can involve the use of chemical crosslinking molecules such as the use of heterobifunctional crosslinking agents such as succinimidyl esters such as 3-(2-pyridyldithio)propionate or succinimidyl acetylthioacetate (Molecular Probes Inc. Handbook, Chapter 5, section 5.3).

Further techniques which may have utility in the conjugation of the antigenic fragment to the Fc fragment would include the techniques described in published International Patent Applications No WO 94/04690 and WO 96/27011.

Conjugation may further be achieved by genetic means through the use of recombinant DNA techniques that are well know in the art such as those set forth in the teachings of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1 , pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989) and F. M. Ausubel et.al. Current Protocols in Molecular Biology, Eds. J.Wiley Press (2006) the relevant portions of which are incorporated herein by reference.

The antigenic peptide which is provided within the immunoconjugate of the present invention is capable of inducing an immune response in an individual to which the immunoconjugate is administered. In certain embodiments, the antigenic peptide is derived from a pathogen or a pathogen product which causes an infectious disease, such that the resulting immune response which is mediated following administration of the immunoconjugate to a subject provides protective immunity against the infectious agent from which the antigen or antigenic fragment is derived. In certain embodiments, the antigenic polypeptide is derived from a protein implicated in a pathogenic process, said pathogenic process causing illness or disease in a subject.

In certain embodiments, the antigenic polypeptide is derived from a secreted product or other infectious agent which is derived from a pathogenic organism. In particular, the secreted product may be derived from a bacterial pathogen and is selected from the group comprising, but not limited to; leukocidins, streptolysin S, streptolysin O, NADase, hyaluronidase, streptokinases, and pyrogenic exotoxins.

In certain embodiments, the antigenic fragment is derived from an Influenza virus which can cause infection in humans. In certain embodiments, the antigenic fragment is haemagglutinin (HA or H) or a fragment thereof derived from an infectious strain of type A influenza virus. In certain embodiments, the antigenic fragment is the HA component of a type A pandemic influenza virus, for example, but not limited to HA1 , HA2, HA3, HA5, HA7 or HA9. In certain embodiments the type A influenza virus comprises haemagglutinin and at least one further influenza antigen including, for example, neuraminidase.

In another embodiment the infectious disease is hepatitis, in particular that caused by type C hepatitis virus. In yet another embodiment the infectious disease is hepatitis, in particular that caused by type B hepatitis virus. In a yet further embodiment the infectious disease is AIDS, in particular that caused by the HIV virus.

In certain embodiments, the antigenic fragment may be derived from a pathogen which mediates an immune response following infection of a host. Such a pathogen would in particular be of the group referred to as an 'infectious agent' or an 'infectious disease' and may be a viral infectious diseases selected from, but not limited to, the group comprising; influenza, rhinovirus and common cold, corona virus such as severe acute respiratory syndrome (SARS) coronavirus, HIV, human paillomavirus, smallpox, rabies, rubella, hepatitis, herpes simplex virus, herpes zoster virus, viral meningitis, yellow fever, west Nile disease, chicken pox (varicella), and foot and mouth disease virus, or bacterial infectious diseases selected from, but not limited to the group comprising; tuberculosis, typhoid, anthrax, bacterial meningitis, cholera, diphtheria, gonorrhoea, legionellosis, leptispirosis, listeriosis, MRSA infection, and pertussis, or parasitic infectious diseases selected from, but not limited to the group comprising; leishmaniasis, malaria and trypanosomiasis, fungal infectious diseases such as; tinea pedis, candidiasis, and prion infectious diseases such as bovine spongiform encephalopathy and Creutzfeldt- Jakob disease.

In a yet further embodiment, the antigenic fragment may be derived from a host protein that is responsible for pathogenesis. Such a protein would include but not be limited to the amyloid proteins that cause pathogenesis in Alzheimer's disease, the nicotinic acid receptor implicated in nicotine addiction and the cholesterol transferase CTEP implicated in the pathogenesis of atherosclerosis.

Although the immunoconjugates of the invention are provided in monomeric form, in certain embodiments, the immunoconjugates may be provided as a dimeric fusion molecule following dimerisation of two immunoconjugates. The resulting dimer may be a homodimer comprised of 2 immunoconjugates having identical antigenic peptides. Alternatively, the dimmer may be formed from 2 immunoconjugates having different antigenic peptides. Where different antigenic peptides are exhibited by the immunoconjugates, these antigenic peptides may be derived from different pathogenic organisms, or may be derived from different target sites of the same pathogenic organisms.

In certain embodiments of the invention, the immunoconjugates may be provided as multimeric molecules. Such multivalent immunoconjugates may be formed using Fc binding regions which are derived from Fc regions, or portions thereof of antibodies which are usually present in a multivalent form, specifically antibodies of the class IgM (pentameric structure) or IgA (dimeric structure). Where multimeric immunoconjugate molecules are formed, the immunoconjugates may comprise similar or different antigenic peptides.

In certain embodiments, the immunoconjugates may have conjugated thereto, further molecules or compounds which may have utility in mediating an immune response.

Production of the polypeptide immunoconiugate of the present invention Expression, isolation and purification of the polypeptides the invention may be accomplished by any suitable technique, including but not limited to the following:

Expression Systems

Expression vectors comprising DNA may be used to prepare the polypeptide immunoconjugate of the present invention encoded by DNA. A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide immunoconjugate of the present invention, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture. The skilled man will recognise that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is intracellular, membrane-bound or a soluble form that is secreted from the host cell.

Any suitable expression system may be employed. The expression vectors used could include a DNA sequence encoding the polypeptide immunoconjugate or a functional fragment thereof operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, avian, microbial, viral, or insect gene.

In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the nucleic acid sequence encoding the immunoconjugate of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, but allows secretion of polypeptide from the cell.

Suitable host cells for expression of the polypeptides of the invention include; prokaryotes, higher eukaryotic cells and yeast. Prokaryotic cells, mammalian cells, and in particular Chinese hamster ovary (CHO) cells are particularly preferred for use as host cells.

Appropriate cloning and expression vectors for use and appropriate mammalian, yeast, fungal, procaryotic and insect cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Lab. Manual, Elsevier, New York, (1986) (ISBN 0444904018) and Current Protocols in Molecular Biology, Eds. F. M. Ausubel et.al., J.Wiley Press (2006), the disclosures of which are incorporated herein by reference.

Prokaryotic Systems

Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example, E.coli, B.subtilis, S.typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.

DNA encoding the polypeptide immunoconjugate of the present invention may be cloned in-frame into the multiple cloning site of an ordinary bacterial expression vector. Ideally the vector would contain an inducible promoter upstream of the cloning site, such that addition of an inducer leads to high-level production of the recombinant protein at a desired time.

For expression of the recombinant protein, the bacterial cells are propagated in growth medium until reaching a pre-determined optical density. Expression of the recombinant protein is then induced. Purification and refolding may then be performed using techniques which will be well known to the person skilled in the art.

Mammalian or Insect Systems Mammalian cell or insect host cell culture systems may also be employed to express recombinant polypeptides. These cause the produced polypeptide to undergo post-translational modifications, such as glycosylation, and this may result in a greater bio-stability of the protein when administered. However, the inventors have identified that the production of the polypeptides of the present invention in systems such as yeast and baculovirus result in the production of a polypeptide which has not undergone mammalian posttranslational modification. While not being bound by theory, it is hypothesised that such changes to the tertiary structure, caused by the absence of complete post-translational modification could include the absence of a fucose group on the N-glycan linked to the asparagine residue at position 276 of the immunoglobulin which could improve binding affinity to the FcR, and increase opportunity for internalisation.

Baculovirus systems for production of heterologous proteins in insect cells are well known those skilled in the art.

Further, established cell lines of mammalian origin are also known, such as the COS-7 line of monkey kidney cells, and Chinese hamster ovary (CHO) cells.

Established methods for introducing DNA into mammalian cells have been described. Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors may also be used.

Yeast Systems The polypeptide immunoconjugate of the present invention may further be expressed in yeast host cells, preferably from the Saccharomyces (e.g., S. cerevisiae), Pichia and Hansula genuses. Particularly preferred are yeast strains that carry modifications that alter the glycosylation and disulphide bond formation in the proteins expressed. Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. Additional yeast stains and protocols are those developed for the expression of antibodies and antibody fragments which are known to those of skill in the art.

Isolation and Purification

With respect to any type of host cell, as is known to the skilled artisan, procedures for purifying a recombinant polypeptide or fragment will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide or fragment is secreted into the culture medium. Particularly preferred are chromatographic methods developed for the purification of antibodies and antibody fragments which are known to those of skill in the art.

Analogues and derivatives The present invention extends to peptides which are derivates or homologues of the polypeptide immunoconjugate of the present invention. Such peptides may have a sequence which has at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, or 90%, 95%, 98% or 99% homology to the sequence of the polypeptide immunoconjugate of the present invention. In one embodiment of the invention the homologue of the Fc domain of the immunoconjugate binds to FcγRIV or homologues thereof with a binding affinity of a sufficient level to induce the internalisation of the FcR and its bound ligand. If required for purification of the polypeptide immunoconjugate may further contain the protein A and protein G binding regions of the Fc domains or other ligand binding sequences such as a His-tag, FLAG-Tag or GST-tag.

As is well understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for 'conservative variation', such as substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Non-peptide mimetics are further provided within the scope of the invention.

Analogues of, and for use in, the invention as defined herein means a peptide modified by varying the amino acid sequence e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids.

Internalisation Screen

Following the binding of the immunoconjugate of the present invention, the binding is not inhibited by antibodies with specificity to FcγRI (CD64). Binding to FcγRIV can be detected by a variety of methods well know in the art, an example of which is the use of FACs to detect binding to cells transfected with FcγRIV. Suitable cells include 293T cells and CHO cells transfected with cDNA encoding FcγRIV. The invention also includes the internalisation of the immunoconjugate by FcγRIV positive antigen presenting cells to define suitable polypeptide immunoconjugates for use in the methods of the present invention.

Although any suitable antigen presenting cell may be used, dendritic cells are preferably used in the identification of suitable antigenic peptide fragments which may thereafter have utility as the antigenic peptide portion of the immunoconjugates of the present invention. In one embodiment of the invention a fluorescent protein domain is fused to the Fc domain sequences to visualise binding and identify appropriate FcγRIV binding fragments with utility for the polypeptide immunoconjugates of the present invention.

Methods for visualizing internalisation of the FcR/Fc complex include immunofluorescent or immunocytochemical labelling of cells using techniques that are well known to persons skilled in the art.

Treatment / Therapy

The term 'treatment' is used herein to refer to any regimen that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.

Administration The polypeptide immunoconjugate for use in the present invention may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration. The polypeptide immunoconjugate of the present invention may be administered to a patient in need of treatment via any suitable route. Route of administration may include; parenteral (including subcutaneous, intramuscular or intravenous), mucosal (including pulmonary) and oral.

In certain embodiments, the composition is deliverable as an injectable composition. For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.

The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition ISBN 0- 912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7th Edition ISBN 0-683305-72-7 the entire disclosures of which is herein incorporated by reference.

Dose

The polypeptide immunoconjugate of the present invention is preferably administered to an individual in a "therapeutically effective amount", this being an amount sufficient to show benefit to the individual. In the case of infectious disease, benefit would include reduction of infection or disease symptoms. In the case of other diseases, benefit would include reduction of disease symptoms. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

EXAMPLES

Example 1 - Production of immunoconiugates For the production of immunoconjugates containing influenza virus haemagglutinin (HA) as the antigenic fragment, HA genes were amplified from viral standards obtained from the National Institute of Biological Standards and Control, Potters Bar (NIBSC) using PCR and cloned into suitable expression vectors containing the Fc gene fragments as fusion proteins. The external domain of the HA3 gene was amplified from influenza strain A/Bangkok/1/79 (BK79) using the forward 5'- GCGCGGCCATTATGGCCCAAAACCTTCCCGGAAATG-3' and reverse δ'-GCGCGGCCGAGGCGGCCCCAGTCTTTGTATCCTGAC-S' primers and the amplified fragment purified, cut with Sfi (sites underlined) and cloned into the Fc fusion protein vector pAc3cFcHis which contains the human IgGI Fc domain, after amplification in the puc-based vector pAcVSV™ Sfi (SD Chappie and IM Jones J. Biotech. (2002) vol.95, p269-27 and Yao YY J. Infect Disease (2004) vol.190, p91-98). The immunoconjugate thus contained amino acids 17-530 inclusive of the BK79 HA3 and the C2/3 (CH2/CH3) domains of human IgGI and was rechecked by sequencing of the final plasmid. Immunoconjugates also contained alanine or glycine amino acid residues which were substituted in place of serine residues at position 298 of the CH2 domain. The fusion protein was expressed in insect cells by construction of recombinant baculovirus vectors using the rapid recombination method of co-transfection of plasmid with linearised baculovirus AcMNPV DNA (Yao YY J Infect. Disease (2004) vol.190, p91-98). Recombinant baculoviruses were titrated on Sf9 cells in multi-well plates and bulk preparations were done in static cultures using serum free Insect Express media (Invitrogen). The immunoconjugates from these static cultures were also used to check protein expression and FcR binding and internalisation using immunohistochemical staining with HA antisera obtained from NIBSC.

Alternatively the commercial BAC-TO-BAC™ system (Invitrogen) was also used. Immunoconjugates were expressed in HIGHFIVE™ or Sf9 cells using 5L Wave Bioreactor vessels (www.wavebiotech.com) and purified for the preparation of vaccines for efficacy testing by using standard affinity chromatography methods and columns ( GE Healthcare, UK).

Similarly immunoconjugates containing the HA from the H5 avian influenza virus AΛ/ietnam/1194/04 as the antigenic peptide component were made by PCR amplification of the external domain using the following forward 5'-

GCGCGGCCATTATGGCCAAGATCAGATTTGCATTGG-3' and reverse δ'-GCGCGGCCGAGGCGGCCTTGGTAAATTCCTATTG-S' primers and were cloned into the FcR vectors for bulk preparation by baculovirus expression in Sf9 cells described as above. The immunoconjugate contained amino acids 17-530 inclusive of the H5 HA and the C2/3 domains of human IgGI and was rechecked by sequencing of the final plasmid.

In a further example, the entire gp120 fragment of the HIV env gene or the outer domain fragment with the deletion of the immunodominant inner domain were cloned into the FcR vector described above in order to provide an immunoconjugate which comprised either the full length gp120 protein or a truncated version thereof conjugated to the CH2/CH3 domains of human IgGL

Example 2- FcyRIV binding studies

Immunoconjugates were further selected based upon their ability to bind to the FcγRIV receptor. Monocytic cells (DCs) expressing FcγRIV were isolated from mouse peripheral blood cells using antibody against FcγRIV attached to magnetic beads. Immunoconjugates were incubated for 15 to 30 minutes at room temperature with the monocytes in RPMI supplemented with 2% foetal calf serum, in the presence or absence of anti-CD64 antibodies. Cells were fixed in 0.1 % glutaraldehyde and permeabilised using 0.3-0.5% non-ionic detergent such as Triton X100 or Nonidet P40. The immunoconjugate was visualised by immunofluorescence using rabbit antisera against the appropriate HA molecule followed by FITC-labelled goat anti-rabbit second layer. Binding was assessed by observation using a NIKON confocal microscope. Binding was not blocked by the addition of anti-CD64 antibodies. The confocal microscope can also be used to determine FcR internalisation following immunoconjugate binding.

Figures 5 and 6 shows the binding of HA-Fc and gp120-Fc immunoconjugates to FcR on human THP1 cell line (Figure 5) and further to FcRs on mouse RAW (Figure 6) cell lines expressing FcγRIV and

FcγRIII as assayed by flow cytometric analysis. Binding was blocked by specific antibodies, but not by competition with mannan, methyl glycoside or lamanarin or all three as shown by the results detailed in Figure 7. Mutation of leucine residues 234 and 235 to valine and alanine residues respectively of the C2/C3 IgGI domain provided for the production of a variant immunoconjugate (for example HA-FcVA as shown in Figure 5 and Figure 6 or gp120-FcVA as shown in Figure 5 and Figure 6). This variant immunoconjugate was shown to have significantly reduced binding to Fc Receptors, as shown by the results of graphs A and B of Figures 5 and 6.

Example 3 - lmmunogenicity of lmmunoconiugates Mice were immunised with the HA-Fc and gp120-Fc immunoconjugates in the absence of any adjuvants and the production of antibodies was assayed by ELISA. Further, the haemagglutinin inhibition (HAI) titres of the antisera using both chicken and turkey erythrocytes was also obtained for the HA-Fc immunoconjugate. Figure 8 shows the significant immunity obtained with the immunoconjugates (group 3) even compared to antisera obtained from mice that were infected with live virus as a positive control (group 4). Immunoconjugates missing the Fc domain (group 1 ) or containing the valine/alanine Fc variant (group 4) showed poor immunity. Similar results were obtained with the gp120 immunoconjugates even when only the poorly immunogenic outer domain of gp120 was used, demonstrating clearly that the fusion to the Fc fragment could significantly enhance the immunogenicity of previously silent antigenic domains.

Example 4 - Protection against heterologous virus (drift of viral sequence by genetic mutation)

Influenza viruses frequently undergo events such as antigenic drift which result in amino acid changes. In order to provide optimum protection, would be highly desirable to provide an immunoconjugate which comprises and antigenic fragment which is derived from an influenza virus which provides protective immunity against further strains of influenza virus which have undergone drift events. Immunoconjugates of HA3 derived from the A/Bangkok/1/79 virus coupled to the Fc domain from IgGI immunoglobulin were used to immunise Balb/c mice, which were then challenged with a heterologous virus AA/ictoria/75 (H3N2) which was contains strain mutations that result in 3 drift events separation from the vaccine strain. The haemagglutinin inhibition (HAI) titres of the antisera induced in the immunized animals was assayed using both chicken and turkey erythrocytes. The ability of the immunoconjugate vaccine to prevent both weight loss and reduce viral load in the lungs was used to assess the level of protection in the immunised animals. Animals were vaccinated at day 0, 13 and 27 with 5ug of immunoconjugate without any adjuvant and challenged at day 41 with a non-lethal dose of infectious heterologous virus. The immunised animals showed both a marked reduction in weight loss (Figure 3) and a 3-fold reduction in lung viral titres showing that the immunoconjugate vaccines protected the animals against viral infection with a heterologous virus.

Similar results were also obtained in relation to the protection of vaccinated mice against challenge with homologous influenza strains. The immunity elicited by the immunoconjugate of this invention is even more surprising as it gives protection not just against heterologous strains that are three drift events apart but it does so at a third of the usual dose given to elicit protection against homologous strains in the annual influenza vaccine. It thus appears that the ability of the Fc- immunoconjugates to induce both cellular and humoral immune responses is sufficient to protect against influenza even in the case of stain variations.

Example 5 - Protection against a pandemic virus (avian influenza) Immunoconjugates of HA5 derived from the AΛ/ietnam/1194/2004 (H5N1 ) avian influenza virus coupled to the Fc domain from IgGI immunoglobulins were used to immunize Balb/c mice which were then challenged with a homologous virus containing an assortment of the AA/ietnam/1194/2004 HA5 with a PR8 virus (NIBRG-14).

Previous work in this mouse model has shown that HA5 subunit vaccines do not protect against infection unless used at high doses in the presence of adjuvants. The haemagglutinin inhibition (HAI) titres of the antisera induced in the immunized animals was assayed using chicken erythrocytes. The ability of the immunoconjugate vaccine to prevent both weight loss and reduce viral load in the lungs was used to assess the level of protection in the immunized animals. Animals were vaccinated at day 0, 14 and 28 with 15ug of immunoconjugate without any adjuvant and challenged at day 43 with a lethal dose of infectious virus carrying the H5 gene from the avian pandemic strain. The immunised animals showed marked protection against infection with a 67% survival rate and a significant reduction of viral titres in the lungs of the immunised animals, including the absence of detectable virus in some of the survivors. Again, the immunised animals (Figure 4) showed no detectable HAI titres before challenge but the immunised animals did show HAI titres against H5 after viral challenge albeit lower than the 4-fold increase in titre required for the licensing of the annual influenza vaccine. Use of the immunoconjugates of this invention resulted in significant survival of the vaccinated animals when challenged with lethal avian influenza strains (Figure 9).

It thus appears that the ability of the Fc immunoconjugates to induce a cellular immune response is sufficient to protect against avian influenza and the use of rapid recombinant DNA technology to produce the immunoconjugates should have particular utility in the production of a vaccine against an emergent pandemic virus. All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. A polypeptide immunoconjugate comprising at least one antigenic peptide or fragment thereof complexed to an FcR binding peptide capable of binding to the FcγRIV receptor or a homologue thereof, wherein the

FcR binding peptide binds to the FcγRIV receptor or a homologue thereof with a binding affinity sufficient to cause internalisation of the bound FcR.

2. A polypeptide immunoconjugate as claimed in claim 1 wherein the FcR binding peptide comprises the amino acid sequence of the CH2 (C2) constant domain derived from the Fc portion of the heavy chain of a human immunoglobulin.

3. A polypeptide immunoconjugate as claimed in claim 2 wherein the FcR binding peptide is comprised of the amino acid residues which comprise the CH2 constant domain and an associated hinge region of an immunoglobulin of the IgG subclass.

4. A polypeptide immunoconjugate as claimed in claim 3 wherein the amino acid sequence of the CH2 domain is genetically modified in the region of the amino acid residue at position 297 in order to increase the binding flexibility of the glycan attached to the residue equivalent to position 297.

5. A polypeptide as claimed in claim 4 wherein the modification to the amino acid residue of residue 297 relates is the substitution of the serine residue at the position equivalent to position 298 to an alanine residue or a glycine residue.

6. A polypeptide as claimed in claim 4 wherein the modification in the region of position 297 relates to the removal of a fucose sugar side chain from the residue at the position equivalent to 297.

7. A polypeptide immunoconjugate as claimed in claims 1 to 6 wherein a linker sequence is provided between the antigenic peptide and the FcR binding peptide.

8. A polypeptide immunoconjugate as claimed in claims 1 to 7 wherein the antigenic peptide is derived from a pathogenic organism or from a protein that is causative of a pathogenic process.

9. A polypeptide immunoconjugate as claimed in claim 8 wherein the pathogenic organism is selected from the group consisting of; a bacterial pathogen, a viral pathogen, a fungal pathogen and a parasitic organism.

10. A polypeptide immunoconjugate as claimed in claim 9 wherein the bacterial pathogen is selected from the group consisting of; tuberculosis, typhoid, anthrax, bacterial meningitis, cholera, diphtheria, gonorrhoea, legionellosis, leptispirosis, listeriosis, MRSA infection and pertussis.

11. A polypeptide immunoconjugate as claimed in claim 9 wherein the viral pathogen is selected from the group consisting of; influenza, rhinovirus, coronavirus, lentivirus, flavivirus, papillomavirus, smallpox, rabies, rubella, dengue hepatitis, herpes simplex virus, herpes zoster virus, viral meningitis, yellow fever, west Nile disease, chicken pox (varicella), and foot and mouth disease virus (FMDV).

12. The polypeptide immunoconjugate as claimed in claim 9 wherein the fungal organism is selected from the group consisting of; aspergillus, Candida, Cryptococcus, histoplasma, coccidioides, blastomyces and penicillium.

13. The polypeptide immunoconjugate as claimed in claim 9 wherein the parasitic organism is selected from the group consisting of; leishmaniasis, malaria and trypanosomiasis.

14. The polypeptide immunoconjugate of any one of claims 1 to 6 wherein the antigenic peptide or fragment is derived from a protein involved in a disease process wherein presentation of the antigenic peptide or a fragment thereof to the cells of the immune system is sufficient to mediate an immune response in the host sufficient to prevent or treat said disease process.

15. The polypeptide immunoconjugate of claim 14 wherein the antigenic peptide may be derived from a protein which is causative of a non-pathogenic inflammatory condition or immune mediate disease selected from the group consisting of; arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), atherosclerosis, stroke, psoriasis, cardiac inflammation, allograft rejection, acute and chronic graft versus host disease, tissue damage resulting from insult or injury, Alzheimer's disease (AD), beta amyloid peptide(s), amyloid precursor protein (APP), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

16. The polypeptide immunoconjugate of any one of the preceding claims wherein the FcR binding peptide is an amino acid sequence derived from the constant domain of the heavy chain of an immunoglobulin.

17. The polypeptide immunoconjugate of claim 16 wherein the FcR binding peptide is an amino acid sequence derived from an immunoglobulin of the isotype IgG.

18. The polypeptide immunoconjugate of claim 17, wherein the FcR binding peptide is derived from an immunoglobulin of the subclass IgGI or lgG3.

19. A method for mediating an immune response against an antigenic peptide in a mammalian host, the method comprising the steps of:

- providing a polypeptide immunoconjugate comprising at least one antigenic peptide or a fragment thereof complexed to an FcR binding peptide capable of binding the FcγRIV receptor or a homologue thereof with an affinity sufficient to cause internalisation of the bound FcR, and

- administering a therapeutically effective amount of said composition to a subject in which the induction of an immune response against said antigenic fragment is desired.

20. The method as claimed in claim 19 wherein the FcR binding peptide comprises an amino acid sequence defining the CH2 (C2) constant domain and the associated hinge region derived from an the Fc portion of the heavy chain of a human immunoglobulin.

21. The method as claimed in claims 19 or 21 wherein a linker moiety or spacer may be provided between the antigenic peptide and the FcR binding peptide components of the immunoconjugate.

22. The method as claimed in claim 19 wherein the antigenic fragment is derived from a pathogenic organism, wherein said fragment is sufficient to allow an immune response to be mediated in a subject to whom the immunoconjugate is administered sufficient to provide protective immunity against the pathogen from which the antigenic fragment is derived.

23. The method as claimed in claim 22 wherein the pathogenic organism is selected from the group consisting of: an infectious disease, a bacterial pathogen, a viral pathogen, a fungal pathogen, and a parasitic pathogen.

24. The method of claim 23 wherein the antigenic fragment is derived from a viral infectious disease selected from the group consisting of; influenza, rhinovirus, coronavirus, flavivirus, papillomavirus, smallpox, rabies, rubella, SARS, hepatitis, herpes simplex virus, herpes zoster virus, viral meningitis, yellow fever, west Nile disease, dengue, chicken pox (varicella), and foot and mouth disease virus (FMDV).

25. The method of claim 23 wherein the antigenic fragment is derived from a bacterial infectious diseases selected from the group consisting of; tuberculosis, typhoid, anthrax, bacterial meningitis, cholera, diphtheria, gonorrhoea, legionellosis, leptispirosis, listeriosis, MRSA infection and pertussis.

26. The method of claim 19 wherein the antigenic peptide or fragment is derived from a protein involved in a disease process wherein presentation of the antigenic peptide or a fragment thereof to the cells of the immune system is sufficient to mediate an immune response in the host sufficient to prevent or treat said disease process.

27. The method of claim 19 wherein the antigenic peptide is derived from a protein which is causative of a non-pathogenic inflammatory condition or immune mediate disease selected from the group consisting of; arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), atherosclerosis, stroke, psoriasis, cardiac inflammation, allograft rejection, acute and chronic graft versus host disease, tissue damage resulting from insult or injury, Alzheimer's disease (AD), beta amyloid peptide(s), amyloid precursor protein (APP), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

28. The method as claimed in claim 19 wherein the antigenic peptide is derived from or is causative of a non-pathogenic inflammatory condition or immune mediate disease selected from the group comprising; arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), atherosclerosis, stroke, psoriasis, cardiac inflammation, allograft rejection, acute and chronic graft versus host disease, tissue damage resulting from insult or injury, Alzheimer's disease (AD), beta amyloid peptide(s), amyloid precursor protein (APP), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

29. The use of the polypeptide immunoconjugate as claimed in any one of claims 1 to 18 for mediating an immune response in a subject.

30. Use as claimed in claim 29 wherein the subject is a mammal.

31. Use as claimed in claim 30 wherein the mammal is a human.

32. The use of the polypeptide immunoconjugate as claimed in any one of claims 1 to 18 for the treatment and / or prophylaxis of an infectious disease.

33. Use according to claim 32 wherein the infectious disease is a viral infectious diseases selected from the group consisiting of; influenza, rhinovirus, coronavirus, HIV, human papillomavirus, smallpox, rabies, rubella, hepatitis, herpes simplex virus, herpes zoster virus, viral meningitis, yellow fever, west Nile disease, chicken pox (varicella), dengue, SARS or foot and mouth disease (FMD).

34. Use according to claim 32 wherein the infectious disease is a bacterial infectious diseases selected from the group consisting of; tuberculosis, typhoid, anthrax, bacterial meningitis, cholera, diphtheria, gonorrhoea, legionellosis, leptispirosis, listeriosis, MRSA infection or pertussis.

35. Use of the polypeptide immunoconjugate according to any one of claims 1 to 18 in the preparation of a medicament for the treatment or prevention of infection of a subject with an infectious disease.

36. Use of the polypeptide immunoconjugate as claimed in any one of claims 1 to 18 wherein the antigenic peptide or fragment is derived from a protein involved in a disease process wherein presentation of the antigenic peptide or a fragment thereof to the cells of the immune system is sufficient to mediate an immune response in the host sufficient to prevent or treat said disease process.

37. Use of the polypeptide immunoconjugate as claimed in claim 36 for the treatment of a non-pathogenic inflammatory condition or immune mediate disease selected from the group consisting of; arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), atherosclerosis, stroke, psoriasis, cardiac inflammation, allograft rejection, acute and chronic graft versus host disease, tissue damage resulting from insult or injury, Alzheimer's disease (AD), beta amyloid peptide(s), amyloid precursor protein (APP), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS- related dementia, encephalitis, stroke and head trauma.

38. A polypeptide immunoconjugate having the primary amino acid sequence ABC, wherein A comprises at least one amino acid which defines at least one antigenic peptide or a fragment thereof, B comprises a linker sequence, and C comprises an amino acid sequence encoding a polypeptide having an FcγRIV binding domain.

39. A polypeptide as claimed in claim 38 wherein C comprises an amino acid sequence derived from the CH2 constant region of the immunoglobulin along with an associated hinge region.

40. A polypeptide immunoconjugate as claimed in claim 39 wherein the amino acid sequence derived from the CH2 domain is modified in the region of position 297 in order to increase the binding flexibility of the glycan attached to the residue equivalent to position 297.

41. A polypeptide as claimed in any one of claims 38 to 40 wherein A defines the N terminal (amino) portion of the polypeptide and C defined the C-terminal (carboxyl) terminal of the polypeptide.