WO2005051414A1 - Use of c4bp core region as a cd40 agonist - Google Patents

Abstract

Use of C4BP Core protein, the C-terminal domain of the alpha chain of C4BP for the manufacture of a medicament for the treatment of chronic autoimmune inflammatory diseases (rheumatoidarthritis, human X-linked hyper-IgM syndrome, combined variable immune deficiency (CVID), post congenital rubella), CD40-negative tumours, B-cell lymphoma, breast tumor. C4BP Core protein can also be used in conjunction with a bacterial, viral, parasitic or tumour antigen where it acts as an adjuvant.

Description

USE OF C4BP CORE REGION AS A CD40 AGONIST

Field of the Invention

The present invention relates to agonists of the cell surface receptor CD40 and their use.

Background to the Invention

CD40

CD40 (GenBank NP_690593) is a cell surface receptor belonging to the tumour necrosis factor-R (TNF-R) family. It was first identified and characterized on B lymphocytes. It has a critical role in T cell-dependent humoral immune responses as was demonstrated by patients with the hyper-IgM syndrome, as well as by gene targeting in mice. However, in recent years it has become clear that CD40 is expressed much more broadly, including expression on monocytes, dendritic cells, endothelial cells, and epithelial cells.

The CD40-ligand (CD40-L/CDl54/gp39) is a member of the TNF family cloned and demonstrated to be a type II integral membrane protein expressed primarily on activated CD4+ T cells. CD40L provides a strong stimulatory signal to B cells, but it is now believed that CD40-CD40-L interactions play a more general role in immune regulation. For reviews on CD40 and CD40L and their signalling, see van Kooten and Banchereau in Journal of Leukocyte Biology (2000) volume 67, pages 2-17 and Schonbeck and Libby in Cell Mol Life Sci. (2001) volume 58, pages 4-43.

Some monoclonal antibodies to the CD40 protein have the useful biological activity of stimulating cells bearing CD40 on their cell surface, and are called agonist anti-CD40 monoclonals. Barr and Heath, in Immunology (2001) volume 102 pages 39-43, showed that those monoclonal antibodies which were strong inhibitors of CD40L binding induced strong proliferative and activation signals to B cells, and conversely those that competed poorly with CD40L for CD40 binding were poor activators of B cells.

However, the use anti-CD40 monoclonal antibodies or of CD40L or of the C4BP alpha chain as agonists of CD40 is limited by a number of factors. Principal among these are the cost of producing the antibody or CD40L or C4BP alpha chain proteins, because they must be expressed in eukaryotic cells. Furthermore, the monoclonal antibodies must be "humanised" to avoid the generation of a neutralising immune response to the monoclonal antibody, as the main agonist monoclonal antibodies to CD40 are derived from mice.

C4 binding protein

C4BP is a high molecular mass (570 kDa) plasma glycoprotein that efficiently inhibits the classical pathway of complement activation and belongs to a gene family of related proteins named the regulators of complement activation (RCA) , which also includes factor H, complement receptors 1 and 2, membrane cofactor protein, and decay accelerating factor.

Each of the proteins of the RCA family binds C4b and/or C3b and is important for the inhibition of the classical and/or alternative pathways of complement activation. All these proteins contain variable numbers of tandemly arranged domains, which are denoted complement control protein (CCP) repeats or short consensus repeats (SCRs) . These domains are cysteine-rich and 60 amino acid residues long, and each is composed of a hydrophobic core that is wrapped by beta- strands, and appear to have a very ancient origin. SCR- containing genes sharing conserved amino acid residues or sequence similarity with SCR regions of human complement genes have been found in insects (Drosophila) , nematodes

{ Caenorhabdi tis elegans) , and sponges ( Geodia cydoniu ) (Hoshino et al. 1993; Ainscough et al . 1998; Blumbach et al . 1998; Pahler et al . 1998) and are reviewed by J. Krushkal, et al . (2000) "Evolutionary Relationships Among Proteins Encoded by the Regulator of Complement Activation Gene Cluster" in Molecular Biology & Evol ution volume 17, pages 1718-1730.

C4BP is synthesized by liver cells and activated monocytes and is upregulated by glucocorticoids and inflammatory cytokines (IFN-γ 11-1, 11-6, and TNF-α) . C4BP is a functional cofactor for Factor I-dependent degradation of C4b and C3b and accelerates decay of C3-convertase . Like other RCA proteins, C4BP has a spider like structure made of seven identical alpha-chains and a single beta-chain. The alpha-chain of C4BP comprises eight tandemly arranged SCR domains of approximately 60 amino acids in length, and a C-terminal region which comprises 57 amino acids. This region is referred to as the C4BP core.

The C4BP alpha chain has a C-terminal core region responsible for assembly of the molecule into a multimer. According to the standard model, the cysteine at position +498 of one C4BP monomer forms a disulphide bond with the cysteine at position +510 of another monomer. A minor form comprising only seven alpha-chains has also been found in human plasma. The natural function of this plasma glycoprotein is to inhibit the classical pathway of complement activation.

The C4BP alpha chain is known to bind to many other molecules, but in each case through one or more SCRs, and never through the C-terminal core domain.

For example, C4BP is known to bind to heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor Vlll/VIIIa via its alpha-chains and with protein S through its beta-chain (Villoutreix BO, Hardig Y, Wallqvist A, Covell DG, Garcia de Frutos P, Dahlback B. (1998) Proteins. 1998 Jun 1; 31 (4) : 391-405) . The C4b binding site of the C4BP alpha chain has been mapped to the first three CCP repeats (Blom, A. M., Webb, J., Villoutreix, B. 0., and

Dahlback, B. (1999) J. Biol . Chem . 274, 19237-19245; Blom, A. M., Foltyn-Zadura, A., Villoutreix, B. 0., and Dahlback, B. (2000) Mol . Immunol . 37, 445-453).

This N-terminal region of C4BP is also important for binding of C4BP to heparin (Blom et al , ibid] , to Bordetella pertussis

(Berggard, K., Johnsson, E., Mooi, F. R., and Lindahl, G.

(1997) Infect . Immun . 65, 3638-3643), and to the M-proteins of Streptococcus pyogenes (Blom, A. M., Berggard, K., Webb, J. H., Lindahl, G., Villoutreix, B. 0., and Dahlback, B. (2000) J. Immunol . 164, 5328-5336). The SCR1 of the alpha chain is principally responsible for binding to Neisseria gonorrhoeae

(Ram S, Cullinane M, Blom AM, Gulati S, McQuillen DP, Boden R, Monks BG, O'Connell C, Elkins C, Pangburn MK, Dahlback B, Rice PA. (2001) Int Immunopharmacol . 1, 423-432).

Similarly, it is to the CCPs of the beta chain of C4BP that protein S is known to bind, rather than to the short C- terminal domain of this subunit . (Webb JH, Villoutreix BO, Dahlback B, Blom AM. (2003) Eur J Biochem. 270, 93-100) .

Libyh et al . , (1997, Blood, 90, 3978-3983), describe a protein multimerisation system which is based on the C-terminal part of the alpha chain of C4BP. The C-terminal part of the C4BP lacks the ability to inhibit the classical pathway of complement activation, but is responsible for polymerisation of C4BP in the cytoplasm of CHO cells producing C4BP. Libyh et al . were able to induce spontaneous multimerisation of associated antibody fragments to create homomultimers of scFv fragments using the C4BP fragment. The C-terminal portion of C4BP used was placed C-terminal to the scFv sequence.

Recently, Mikata et al . have shown that the C4BP alpha chain lacking the core domain remains a very effective inhibitor of complement, if the fragment is oligomerised by presentation on the cell membrane (1998, Transplantation, volume 65, pages 363-368). Indeed, the alternative method of oligomerisation converts the "membrane C4BP" into a potent inhibitor of the alternative pathway of complement activation, whereas native C4BP inhibits only the classical pathway (Mikata et al . 1998, Molecular Immunology, volume 35, pages 537-544). The results in these papers are consistent with the standard view that oligomerisation is the sole function of the core domain of C4BP.

Recently, Brodeur et al . in Immunity, 2003, volume 18, pages 837-848 demonstrated that the alpha chain of human C4BP binds directly to CD40 on human B cells at a site that differs from that used by CD40 ligand. The binding of C4BP induces proliferation, upregulation of CD54 and CD86 expression, and IL4-dependent IgE isotype switching in normal B cells. These observations suggest that C4BP is an activating ligand for CD40, which does not interfere with CD40L signalling and might even be synergistic.

In discussing the work of Brodeur, Clark and Craxton in

Immunity, 2003, volume 18 pages 724-725 state that: "It will also be important to know which SCRs in C4BP binds to CD40..". The potential of SCR1 or SCR2 binding to CD40 (and bringing bacterial products closer to pattern recognition receptors) is discussed, but the possibility of the core protein binding to CD40 is not.

Disclosure of the Invention

The present invention provides a method of agonising CD40 on the surface of a cell, which method comprises bringing the cell into contact with an effective amount of a C4BP core protein. The method may be performed in vivo or in vi tro .

The invention provides a method of treating a subject which method comprises administering to the subject an effective amount of a C4BP core protein.

The subject may be a human or animal subject. The subject may be one with a disease associated with a defect in CD40L activity or expression, or a subject with a condition which will benefit from an enhanced B-cell response. Furthermore, the subject may be a patient receiving immunotherapy, either prophylactic or therapeutic, with an antigen where the C4BP core protein acts as an adjuvant.

The invention also provides a C4BP core protein for use in the therapies and treatments mentioned herein.

The invention further provides the use of a C4BP core protein for the manufacture of a medicament for the therapies mentioned herein.

The invention further provides the use of C4BP as an adjuvant, and compositions comprising an immunogen and C4BP, and their use as vaccines and immunotherapeutic compositions. In another aspect, the invention provides a method of vaccinating a subject with an antigen which method comprises administering to the subject an effective amount of a vector encoding C4BP together with an antigen. The antigen may also be delivered in the form of a gene therapy vector, either on the same vector or on a separate vector. The vector may be in the form of an expression construct integrated into the genome of an attenuated organism.

Description of the Drawings

Figure 1 shows an alignment of C4BP core proteins.

Detailed Description of the Invention

Core protein of C4BP alpha chain.

This is referred to herein as the "C4BP core protein" or "core protein". The terms are used interchangeably. This protein may be a mammalian C4BP core protein or a fragment thereof capable of forming multimers, or a synthetic variant thereof capable of forming multimers and capable of binding to CD40.

The sequences of a number of mammalian C4BP proteins are available in the art. These include human C4BP core protein (SEQ ID N0:1). There are a number of homologues of human C4BP core protein available in the art. There are two types of homologue: orthologues and paralogues. Orthologues are defined as homologous genes in different organisms, i.e. the genes share a common ancestor coincident with the speciation event that generated them. Paralogues are defined as homologous genes in the same organism derived from a gene, chromosome or genome duplication, i.e. the common ancestor of the genes occurred since the last speciation event. For example, a search of GenBank indicates mammalian C4BP core homologue proteins in species including rabbit, rat, mouse and bovine origin (SEQ ID NO:2-5 respectively). Paralogues have been identified in pig (ApoR) , guinea pig (AM67) and mouse (ZP3) ; shown as SEQ ID NO: 6-8 respectively.

An alignment of SEQ ID NOs:l-8 is shown as Figure 1. It can be seen that all eight sequences have a high degree of similarity, though with a greater degree of variation at the C-terminal end. Further C4BP core proteins may be identified by searching databases of DNA or protein sequences, using commonly available search programs such as BLAST.

Where a C4BP protein from a desired mammalian source is not available in a database, it may be obtained using routine cloning methodology well established in the art. In essence, such techniques comprise using nucleic acid encoding one of the available C4BP core proteins as a probe to recover and to determine the sequence of the C4BP core proteins from other species of interest. A wide variety of techniques are available for this, for example PCR amplification and cloning of the gene using a suitable source of mRNA (e.g. from an embryo or an actively dividing differentiated or tumour cell) , or by methods comprising obtaining a cDNA library from the mammal, e.g. a cDNA library from one of the above-mentioned sources, probing said library with a known C4BP nucleic acid under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C), and recovering a cDNA encoding all or part of the C4BP protein of that mammal. Where a partial cDNA is obtained, the full length coding sequence may be determined by primer extension techniques.

A fragment of a C4BP core protein capable of forming multimers may comprise at least 47 amino acids, preferably at least 50 amino acids. The ability of the fragment to form multimers may be tested by expressing the fragment in a prokaryotic host cell according to the invention, and recovering the C4BP fragment under conditions which result in multimerisation of the full 57 amino acid C4BP core, and determining whether the fragment also forms multimers. Desirably a fragment of C4BP core comprises at least residues 6-52 of SEQ ID NO : 1 or the corresponding residues of its homologues.

The human C4BP core protein of SEQ ID NO:l corresponds to amino acids +493 to +549 of full length C4BP protein sequence. A fragment of this known in the art to form multimers corresponds to amino acids +498 to +549 of C4BP core protein. For the treatment or therapy of a subject which is a human, the human C4BP core protein or above-described fragments thereof are preferred.

Variants of C4BP core and fragments capable of forming multimers, which variants likewise retain the ability to form multimers (which may be determined as described above for fragments) may also be used. The variant will preferably have at least 70%, more preferably at least 80%, even more preferably at least 90%, for example at least 95% or most preferably at least 98% sequence identity to a wild type mammalian C4BP core or a multimer-forming fragment thereof. In one aspect, the C4BP core will be a core which includes the two cysteine residues which appear at positions 6 and 18 of SEQ ID Nos:l-3 and 5-8. Desirably, the variant will retain the relative spacing between these two residues.

The above-specified degree of identity will be to any one of SEQ ID N0s:l-8 or a multimer-forming fragment thereof. Most preferably the specified degree of identity will be to SEQ ID N0:1 or a multimer-forming fragment thereof.

The degree of sequence identity may be determined by the algorithm GAP, part of the "Wisconsin package" of algorithms widely used in the art and available from Accelrys (formerly Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences in a way that maximises the number of matches and minimises the number of gaps. GAP is useful for alignment of short closely related sequences of similar length, and thus is suitable for determining if a sequence meets the identity levels mentioned above. GAP may be used with default parameters.

Synthetic variants of a mammalian C4BP core protein include those with one or more amino acid substitutions, deletions or insertions or additions to the C- or N-termini. Substitutions are particularly envisaged. Substitutions include conservative substitutions. Examples of conservative substitutions include those set out in the following table, where amino acids on the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Examples of fragments and variants of the C4BP core protein which may be made and tested for their ability to form multimers thus include SEQ ID NOs : 9 to 16, shown in Table 1 below:

A=SEQ ID NO:; B= sequence, C= % identity, calculated by reference to a fragment of SEQ ID NO : 1 of the same length.

Where deletions of the sequence are made, apart from N- or C- terminal truncations, these will preferably be limited to no more than one, two or three deletions which may be contiguous or non-contiguous.

Where insertions are made, or N- or C-terminal extensions to the core protein sequence, these will also be desirably limited in number so that the size of the core protein does not exceed the length of the wild type sequence by more than 20, preferably by more than 15, more preferably no more than 10, amino acids. Thus in the case of SEQ ID NO:l, the core protein, when modified by insertion or elongation, will desirably be no more than 77 amino acids in length.

Production of C4BP core protein

C4BP may be made by any convenient route known as such in the art of protein expression and production. A particularly preferred means of production is in a prokaryotic host cell, such as E. coli . Plasmids encoding C4BP may be introduced into the host cells using conventional transformation techniques, and the cells cultured under conditions to facilitate the production of the fusion protein. Where an inducible promoter is used, the cells may initially be cultured in the absence of the inducer, which may then be added once the cells are growing at a higher density in order to maximise recovery of protein.

Cell culture conditions are widely known in the art and may be used in accordance with procedures known as such.

C4BP is a secreted protein in mammals, and these are known in the art to be particularly difficult to produce in a correctly folded form in prokaryotes. Proteins with disulphide bridges are particularly problematic, as are those that require oligo- merisation. Disulphide bonds are not normally produced in the reducing environment of the bacterial cytoplasm, and when they can form, they can stabilise mis-folded or aggregated forms of the protein.

Usually, recombinant proteins expressed in prokaryotes are aggregated inside inclusion bodies within the host prokaryotic cell. These are discrete particles or globules separate from the rest of the cell which contain the expressed proteins usually in an agglomerated or inactive form. The presence of the expressed protein in the inclusion bodies makes it difficult to recover the protein in active soluble form as the necessary refolding techniques are techniques are inefficient and costly. Proteins purified from inclusion bodies have to be laboriously manipulated, denatured and refolded to obtain active functional proteins at relatively poor yields.

With regard to expressing C4BP core fusion proteins in prokaryotic cells, other considerations have also to be taken into account. Firstly, each core monomer retains two cysteine residues, and according to the model of C4BP multimers accepted in the art, these cysteines are required to form inter-molecular disulphide bonds during the assembly of multimers. The reducing environment of the prokaryotic cytosol such as the bacterial cytosol would be expected to prevent the formation of C4BP core multimers by reducing these disulphide bonds .

Secondly, multimers are assembled during passage through the eukaryotic secretion apparatus, which is known to assist protein folding in ways not available in prokaryotes (e.g. the presence of protein disulphide isomerase and unique chaperones) . Thirdly, even under conditions where relatively small yields were obtained in eukaryotic cells (micrograms per millilitre) , this secretory pathway is unable to produce homogenous protein.

We have found that the yield of protein in cell cultures of the invention can be relatively high, for example greater than 2 mg/1 of culture, such as greater than 5 mg/1 of culture, preferably greater than 10 mg/1 of culture, such as greater than 20 mg/1 culture, and even more preferably greater than 100 mg/1 culture.

Once the cells have been grown to allow for production of the protein, the protein may be recovered from the cells. Because we have found that surprisingly, the protein remains soluble, the cells will usually be spun down and lysed by sonication, for example, which keeps the protein fraction soluble and allows this fraction to remain in the supernatant following a further higher speed (e.g. 15,000 rpm for 1 hour) centrifugation .

The core protein in the supernatant protein fraction may be purified further by any suitable combination of standard protein chromatography techniques. We have used ion-exchange chromatography followed by gel filtration chromatography. Other chromatographic techniques, such as affinity chromatography, may also be used.

In one embodiment, we have found that heating the supernatant sample either after centrifugation of the lysate, or after any of the other purification steps will assist recovery of the protein. The sample may be heated to about 70 - 80 °C for a period of about 10 to 30 minutes.

Depending on the intended uses of the protein, the protein may be subjected to further purification steps, for example dialysis, or to concentration steps, for example freeze drying.

Treatment of a subject

The method of the invention may be practiced for the treatment of a subject involving any condition or therapy in which it is desired to activate the CD40 molecule.

In one aspect, the condition may be a defect in the immune system. For example, Mauri et al (Nat Med. (2000) volume 6 pages 673-679) show that an agonistic monoclonal antibody against CD40 can potentially be used to treat chronic autoimmune inflammatory processes, such as those observed in animal models of rheumatoid arthritis. Thus the present invention provides a C4BP core protein for use in a method for the treatment of rheumatoid arthritis.

US Patent 6,106,832 describes the use of CD40 ligands in a method of treatment of an individual that has a syndrome in which the interaction of T cells and B cells is affected, and in particular conditions associated with elevated levels of IgM such as human X-linked hyper-IgM syndrome. This syndrome is characterized by an elevated level of serum IgM and diminished (virtually undetectable) levels of other isotypes of immunoglobulins .

Elevated levels of serum IgM occur in other syndromes, including combined variable immune deficiency (CVID) and post congenital rubella.

Thus the present invention provides a C4BP core protein for use in a method for the treatment of a condition selected from the group of human X-linked hyper-IgM syndrome, combined variable immune deficiency (CVID) and post congenital rubella.

CD40 agonist ligands are also known to be of use in the treatment of a variety of cancers, including solid tumours.

For example, Todryk et al (J Immunol Methods. (2001) volume 248; 139-47) describe the treatment of a range of CD40- negative tumours with anti-CD40 monoclonal antibody. Thus the present invention provides a C4BP core protein for use in a method for the treatment of a CD40-negative tumour. This treatment was particularly effective with two different colorectal tumours .

Honeychurch et al (Blood 2003 volume 102; 1449-1457) describe the simultaneous use of agonistic anti-CD40 monoclonal antibody with other treatment modalities, in particular radiation, in the treatment of models of B-cell lymphomas. The authors suggest that the external irradiation induced an initial kill of lymphoma cells, probably by apoptosis, which releases tumour antigens and slows the progression of the malignancy to allow generation of a curative cytotoxic T lymphocyte (CTL) response promoted by the anti-CD40 mAb. Combining irradiation with immunomodulatory mAb appears to provide a powerful new approach to the management of cancer. Accordingly the C4BP core protein may be used in a combination treatment in which it is administered simultaneously or sequentially with radiation therapy to a patient undergoing treatment of B-cell lymphoma. The present invention also provides a C4BP core protein for use in the treatment of a patient, wherein said patient is one who is receiving, or has received within the previous 96, preferably 48, more preferably 24 hours at least one dose of radiation therapy for the treatment of a B-cell lymphoma.

French et al , (Nat Med. 1999 May; 5 (5); 548-536) show that CD40 antibody evokes a cytotoxic T-cell response to eradicate lymphoma and bypass T-cell help. The authors demonstrated that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurred. Thus the invention provides the C4BP core protein for use in a method of treating a lymphoma.

Turner et al , (J Immunol. 2001 volume 166; 89-94) teach that an anti CD40 antibody induces anti-tumour and antimetastatic effects by activation of NK cells which enhances production of IL-12 and interferon gamma in vivo . This demonstrates that a CD40 agonist has a general anti-tumour effect and antimetastatic effect. Accordingly the C4BP core protein may be used in a method of treating a primary or secondary tumour.

Tumours which may be treated in accordance with the invention include melanomas, colorectal tumours and neuroblastomas .

In particular, breast tumour may be treated. Hirano et al (Blood. (1999) volume 93; 2999-3007) show that a soluble recombinant human CD40 ligand inhibits the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma . Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand.

Thus the invention provides a C4BP for use in a method of treating CD40(+) tumours and in particular a CD40(+) breast cancer, optionally in combination with interferon gamma. The interferon gamma may be administered in combination with or sequentially to the administration of C4BP (or vice versa ) .

The C4BP may also be used as an adjuvant to improve the immune response. For example, Rolph and Kaufmann (J Immunol. 2001

Apr 15; 166 (8); 5115-21) report that CD40 signalling converts a minimally immunogenic antigen into a potent vaccine against the intracellular pathogen Listeria monocytogenes .

Similarly, Ferlin et al (Eur J Immunol. (1998) volume 28; 525- 531) show that in inducing a protective response in Leishmania major-infected BALB/c mice, an anti-CD40 mAb induces a protective Thl response. This is in contrast to the Th2 response which is normally developed after exposure to this parasite .

In addition to the Thl response, Dullforce et al , (Nat Med. 1998 Jan;4(l); 88-91) report that CD40 agonist antibodies enhance a T-cell independent immune response. This is likely to prove beneficial in vaccination of subjects, particularly infants, against encapsulated bacterial pathogens such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis .

Accordingly, the present invention provides a C4BP for use as an adjuvant to enhance the immune response against an antigen. The antigen may be from an infectious agent such as a parasite, bacteria or virus. The antigen may also be a tumour antigen.

Bacterial sources of antigens include antigens from the bacterial agents responsible for bacterial pneumonia, pneumo- cystis pneumonia, meningitis, cholera, tetanus, tuberculosis and leprosy.

Parasitic sources of antigens include malarial parasites, such as Plasmodium.

Viral sources include poxviruses, e.g., cowpox virus and orf virus; herpes viruses, e.g., herpes simplex virus type 1 and 2, B-virus, varicellazoster virus, cytomegalovirus, and Epstein-Barr virus; adenoviruses, e.g., mastadenovirus; papovaviruses, e.g., papillomaviruses such as HPV16, and polyomaviruses such as BK and JC virus; parvoviruses, e.g., adeno-associated virus; reoviruses, e.g., reoviruses 1, 2 and 3; orbiviruses, e.g., Colorado tick fever; rotaviruses, e.g., human rotaviruses; alphaviruses, e.g., Eastern encephalitis virus and Venezuelan encephalitis virus; rubiviruses, e.g., rubella; flaviviruses, e.g., yellow fever virus, Dengue fever viruses, Japanese encephalitis virus, Tick-borne encephalitis virus and hepatitis C virus; coronaviruses, e.g., human coronaviruses; paramyxoviruses, e.g., parainfluenza 1, 2, 3 and 4 and mumps; morbilliviruses, e.g., measles virus; pneumovirus, e.g., respiratory syncytial virus; vesiculoviruses, e.g., vesicular stomatitis virus; lyssaviruses, e.g., rabies virus; orthomyxoviruses, e.g., influenza A and B; bunyaviruses e.g., LaCrosse virus; phleboviruses, e.g., Rift Valley fever virus; nairoviruses, e.g., Congo hemorrhagic fever virus; hepadnaviridae, e.g., hepatitis B; arenaviruses, e.g., 1cm virus, Lasso virus and Junin virus; retroviruses, e.g., HTLV I, HTLV II, HIV-1 and HIV-2; enteroviruses, e.g., polio virus 1,- 2 and 3, coxsackie viruses, echoviruses, human enteroviruses, hepatitis A virus, hepatitis E virus, and Norwalk-virus; rhinoviruses e.g., human rhinovirus; and filoviridae, e.g., Marburg (disease) virus and Ebola virus .

Thus the invention provides a C4BP as an adjuvant for use in combination with an antigen from any of the above-mentioned parasites, bacteria or viruses. The antigen may be in the form of an isolated protein, or mixtures thereof (which may be native or recombinant protein) or it may be present on the organism to which the vaccination is directed, e.g. when a killed or attenuated vaccine is used. By "in combination with an antigen" it is meant the components (C4BP and antigen) are administered at or about the same time, as the two components are unlinked.

Furthermore, the C4BP may be provided by incorporating a nucleic acid encoding the C4BP if a live organism is used for vaccination. Thus,' for example, a BCG (Bacille Calmette Guerin) vaccine strain could be modified so that it produced recombinant C4BP, thereby increasing the immunogenicity of the vaccine. Murray et al . (1996, Proc. Natl. Acad. Sci. volume 93, pages 934-939) have described how to create genetically modified BCG strains secreting cytokines, to the same end.

Tumour antigens include antigens such as one selected from the group consisting of prostate-specific antigen (PSA) , human leukemia-associated antigen, carcinoembryonic antigen (CEA) , the melanoma-specific antigens MAGE-1, and MART-1.

Composi tions

The present invention provides compositions of C4BP core protein, optionally together with an immunogen, such as an immunogen described herein above. This means that core protein and immunogen will be present as separate, unlinked components of the composition.

The composition will in addition comprise a pharmaceutically acceptable carrier. The composition will be prepared according to the intended use and route of administration of the product. Thus the invention provides a composition comprising a product of the invention in multimeric form together with one or more pharmaceutically acceptable carriers or diluents, and the use of such a composition in methods of immunotherapy for treatment or prophylaxis of a human or animal subject.

Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, a fusion protein of the invention optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the composition to be administered may also auxiliary substances such as pH buffering agents and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 19th Edition, 1995.

The composition or formulation to be administered will, in any event, contain a quantity of the active compound (s) in an amount effective to alleviate the symptoms of the subject being treated. Dosage forms or compositions containing active ingredient in the range of 0.25 to 95% with the balance made up from non-toxic carrier may be prepared.

Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. A more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., US Patent No. 3,710,795.

Doses of C4BP will be in the range of 0.2 to 20 mg/kg body weight per day. Doses may be administered at any suitable interval determined by the physician, for example at daily, twice-weekly or weekly intervals.

Where C4BP is administered as an adjuvant, it may also be administered in the dose ranges indicated above. The amount of antigen will be dependent upon the nature of the antigen and may be determined according to current practice for administration of that antigen in conventional vaccine formulations . Vectors

In a further aspect, the C4BP may be administered to a subject in the form a nucleic acid vector encoding the C4BP such that the C4BP is expressed in cells of the subject. Various gene therapy vectors are described in the art, for example in US patents 6,339,068 and 6,228,844, the contents of which are incorporated herein by reference.

Typically, the vector will comprise a nucleic acid sequence encoding C4BP operably linked to a promoter which is active in one or more cell types in a subject. The vector may be a DNA or RNA vector. Different types of vectors are known in the art, and include plasmid vectors and viral vectors. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) , and Rous Sarcoma Virus (RSV) . When the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) can be utilized. A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.

Where viral vectors are used, the expression of C4BP may be driven from a native viral promoter. Alternatively, or in the case of plasmid vectors, the promoter may be any other promoter which will work in a target cell in the subject.

Such promoters may include the mouse metallothionein I gene (Hamer et al . , J. Molec. AppL Genet. 1: 273 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31: 355 (1982); the SV40 early promoter (Benoist et al . , Nature 290: 304 (1981); the Rous sarcoma virus promoter (Gorman et al . , Proc. Natl . Acad. Sci. USA 79: 6777 (1982); and the cytomegalovirus promoter (Foecking et al . , Gene 45: 101 (1980)).

Delivery of gene therapy vectors may be accomplished by administration of "naked" nucleic acid or, in the case of viral vectors, by viral particles (though viral nucleic acid may also be administered as naked nucleic acid.

The vectors may be formulated in the form of a pharmaceutical composition comprising the vector and a suitable carrier. The pharmaceutical compositions according to the invention are prepared by bringing the construct according to the present invention into a form suitable for administration to a subject using a carrier, e.g. a solvent, delivery system, excipient, and additive or auxiliary. Frequently used solvents include sterile water and saline (buffered or not) . Another carrier includes gold particles, which are delivered biolistically (ie., under gas pressure).

The nucleic acid may be encapsulated in a liposome. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol . Infect. Dis. 12 (Suppl. 1): Sβl (1993), and Kim, Drugs 46: 618 (1993).

Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 μm to greater than 10 μm. See, for example, Machy et al . , LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et al., American J. Hosp. Phann. 46: 1576 (1989).

After intravenous administration, conventional liposomes are preferentially phagocytosed into the reticuloendothelial system. However, the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means. Claassen et al., Biochim. Biophys . Acta 802: 428 (1984). In addition, incorporation of glycolipid- or polyethelene glycol- derivatised phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system. Allen et al . , Biochim. Biophys. Acta 1068: 133 (1991); Allen et al . , Biochim. Biohys . Acta 1150: 9 (1993). These Stealth. RTM. liposomes have an increased circulation time and an improved targeting to tumors in animals. (Woodle et al . , Proc. Amer. Assoc. Cancer Res. 33: 2672 1992). Human clinical trials are in progress, including Phase III clinical trials against Kaposi's sarcoma. (Gregoriadis et al., Drugs 45: 15, 1993).

Nucleic acid can be encapsulated within liposomes using standard techniques. A variety of different liposome compositions and methods for synthesis are known to those of skill in the art. See, for example, U.S. Pat. No. 4,844,904, U.S. Pat. No. 5,000,959, U.S. Pat. No. 4,863,740, U.S. Pat. No. 5,589,466, U.S. Pat. No. 5,580,859, and U.S. Pat. No. 4,975,282, all of which are hereby incorporated by reference.

Liposomes can be prepared for targeting to particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For instance, antibodies specific to tumour associated antigens may be incorporated into liposomes, together with gene therapy vectors, to target the liposome more effectively to the tumour cells. See, for example, Zelphati et al . , Antisense Research and Development 3: 323-338 (1993), describing the use "immunoliposomes" containing vectors for human therapy.

In general, the dosage of administered liposome-encapsulated vectors will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Dose ranges for particular formulations can be determined by using a suitable animal model .

As an alternative approach the C4BP vector may be in the form of an expression construct integrated into the genome of an attenuated organism. In this manner, the C4BP will be encoded in the DNA of, and expressed by, attenuated organisms such as BCG or fowlpox or Modified Vaccinia Ankara (MVA) . The C4BP may be expressed from an endogenous promoter of the organism. In this case, the C4BP may be integrated into the genome of the organism by targeted homologous recombination in order to replace a coding sequence of the organism with the coding sequence of C4BP such that the C4BP is driven by the promoter of the original coding sequence. Such a coding sequence may itself be a gene associated with virulence, such that the organism is attenuated by the process which produces the C4BP- expressing organism.

Such organisms may be administered to a subject in accordance with standard procedures known as such in the art of vaccination.

The vectors or attenuated organisms encoding C4BP may further encode an antigen to which it is desired to enhance an immune response in the subject. The vectors or attenuated organisms will usually express the antigen from a separate promoter, which may be the same or different as the promoter which expresses the C4BP.

The present invention is illustrated by the following examples .

Example 1 - Production of db-C4BP

Vector construct .

An expression vector encoding the downstream box peptide sequence MASMNHKGS (Sprengert M.L., Fuchs E. and Porter A.G 1996 "The downstream box: an efficient and independent translation initiation signal in Escherichia coli . " EMBO J. Volume 15, 665-674) fused N-terminal to the 57 amino acid "core" domain of the human C4BP alpha chain was constructed.

Briefly, the C4BP core domain is encoded entirely within a single exon in the human genome, thus allowing it to be amplified directly from human genomic DNA. The oligo- nucleotide primers used were:

AVD102: 5' CCCGCGGATCCGAGACCCCCGAAGGCTGTGA3' ; and

AVD103: 5' CCCCGGAATTCTTATTATAGTTCTTTATCCAAAGTGG3' .

These contained added restriction sites which were used for cloning the amplified DNA fragment. The 183 base-pair fragment obtained on digesting the PCR product with the enzymes BamHI and EcoRI was cloned downstream of the translational enhancer or "downstream box" and the T7 promoter in a plasmid vector. The plasmid was derived from the plasmid pRsetA supplied by Invitrogen, but the fl origin of replication has been replaced by the par locus from the plasmid pSClOl. It thus contains as functional elements: a selectable marker (ampicillin resistance) an origin of replication (derived from the pUC family) and a T7 promoter and a T7 transcription terminator as well as the par locus. The resulting construct was designated plasmid pAVD 77. Table 2 shows the sequence of the translational enhancer and T7 promoter fused to the coding sequence of C4BP (in small print) .

Table 1 - SEQ ID NO: 17

GATCTCGATC CCGCGAAATT AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT

AGAAATAATT TTGTTTAACT TTAAGAAGGA GATATACATA TGGCTAGCAT GAATCACAAA

GGATCCgaga cccccgaagg ctgtgaacaa gtgctcacag gcaaaagact catgcagtgt ctcccaaacc cagaggatgt gaaaatggcc ctggaggtat ataagctgtc tctggaaatt gaacaactgg aactacagag agacagcgca agacaatcca ctttggataa agaactataa taa

The predicted size of the db-C4BP fusion protein is 7491.5 Da.

Transformation and expression.

The vector was transformed into the E. coli strain C41(DE3), a derivative (Bruno Miroux and John E. Walker 1996 "Overproduction of Proteins in Escherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular

Proteins at High Levels." Journal of Molecular Biology Volume 260, 289-298) of BL21(DE3).

One litre of LB-Ampicillin medium was inoculated with the cells, which were incubated at 37 °C with shaking for 3 hours (until OD600 nm reached 0.6) and then it was induced with IPTG (isopropylthiogalactoside) at a final concentration 0.7 mM for 3 hours. The cells were harvested by centrifugation at 4600 rpm for 30 min. The pellet (P) was resuspended with 30 mis Tris 50 mM pH 7, and the cells were broken by sonication using an Emulsiflex apparatus twice (between each treatment, centrifugation at 15000 rpm for 1 hour, the supernatants from each spin (designated SNl and SN2 respectively) were kept and the pellet Pi was resuspended with the same buffer) .

Both supernatants were pooled (60 mis) and were split into two solutions of 30 mis. Each of these 30 ml aliquots of the db- C4BP fusion protein was purified using one of two similar methods: these were identical except that a heating step in one method was replaced by a MonoQ ion-exchange step in the other .

Purification without a heating step

The native db-C4BP was purified from 500 mis of culture by ion-exchange chromatography (DEAE Fast Flow 70, using a column of 13cm in height, and diameter of 2.6cm), using TrisHCl buffer (50mM pH7) and a salt gradient (0M - 1M NaCl) . The fusion protein eluted between 300-400 mM NaCl. Fractions of 7.5 ml each were collected.

Fractions B8 to Bll were pooled and dialyzed against TrisHCl 20 mM pH7. Then this solution was loaded on a ion-exchange column (MonoQ HR 16/10), using Tris buffer (50mM pH7) and a salt gradient (0M - 1M NaCl). Fractions of 2.5 ml were collected. The fusion protein eluted between 500-550 M NaCl.

Fractions A10 to Bl were pooled and the final solution was then concentrated to a volume of 10 mis before being chromatographed on a gel filtration column (S75 26/60) . Fractions of 5 ml were collected. The fusion protein was eluted from this column with a volume of 139 mis buffer (TrisHCl 100 mM pH7, 150 mM NaCl) . The calibration of the column with molecular weight standards implies a molecular weight for this protein similar to albumin (67 kDa) , which in Tris 50 mM + NaCl 150 mM also elutes with a volume of 139 mis, whereas the expected molecular weight of the monomer is 7.491 kDa. This indicates that the fusion protein is oligomeric in structure when purified from the cytosol of E. coli, without any steps being taken to refold it.

Fractions A10 to Bl were pooled (312 μg/ml) , and an aliquot was dialysed against sodium phosphate buffer, 100 mM, pH 7.4.

The protein yield per Litre of culture after purification was 12.4 milligrams.

The CD spectrum was examined and showed the presence of significant secondary structure, consistent with a properly folded protein complex.

Purification of db-C4BP with a heating step

The solution containing the other 30 ml aliquot of db-C4BP was heated at 76°C for 15 minutes and then centrifuged at 20,500 rpm for 1 hour. The supernatant, containing db-C4BP, was purified by ion-exchange chromatography (DEAE Fast Flow 70 mis), using Tris buffer (50mM pH7) and a salt gradient (0M - 1M NaCl). Fractions of 7.5 ml were collected. The fusion protein eluted between 300-400 mM NaCl.

Fractions B8 to Bll were pooled and the final solution was then concentrated to a volume of 10 mis before being chromatographed on a gel filtration column (S-75 26/60) . Fractions of 5 ml were collected. The fusion protein was eluted from this column with a volume of 140mls buffer. The calibration of the column with molecular weight standards implies a molecular weight identical to that of the protein purified without heating (see above) , whereas the expected molecular weight of the monomer is 7.491 kDa. This fusion protein is therefore also oligomeric in structure when 5 purified from the cytosol of E. coli, without any steps being taken to refold it. Furthermore, it remains oligomeric despite being heated to 76°C for 15 minutes in a buffer comprising 50 mM TrisHCl pH7 (i.e. no salt was present).

Fractions All to Bl were pooled (595.5 μg/ml) and an aliquoto was dialysed against sodium phosphate (NaP) buffer 100 mM pH 7.4.

Analysis using circular dichroism showed that the spectrum obtained with the sample which had been subjected to heating was equivalent to that obtained using the unheated sample.₅ This demonstrated that the secondary structure elements of the protein are retained despite heating.

The yield with the heating step was 3.5 milligrams per litre.

The addition of a heating step can significantly simplify the purification of proteins. In the example here, heating o replaced one ion-exchange (MonoQ) step, and nevertheless resulted in a protein of at least equivalent purity.

Example 2 - Binding of C4BP core protein to CD40.

To demonstrate binding of C4BP core to CD40, the methods of Brodeur et al , ibid, who have done all the assays with full-5 length C4BP, may be followed using the C4BP core.

The protein sCD40:Ig (a fusion protein comprising the extracellular portion of CD40 fused to the Ig heavy chain fragment Fc is purchased from, for example, Ancell (Bayview, MN) . One microgram of this fusion protein is coupled to Protein G sepharose beads, and incubated with the C4BP core protein, at a range of concentrations of the latter (from 0.1 microgram, 0.5 micrograms, 1 microgram, 5 micrograms, 10 micrograms, 50 micrograms) in a 100 microlitre volume solution of the tissue culture medium RPMI1640 to which Bovine serum albumin had been added to a final concentration of 0.5% (to block non-specific binding) .

In parallel, positive and negative controls are run. In the positive control, human C4BP protein (containing both the alpha and the beta chain) instead of the C4BP core protein, and at the same concentrations as above.

In the negative control, murine IgG2a is used instead of sCD40:Ig.

The protein complexes are washed four times in the binding solution (RPMI1640 with 0.5% BSA), precipitated one final time and then blotted and probed with rabbit anti-C4BP IgG and anti-rabbit IgG HRP conjugates and detected by enhanced chemiluminescense .

Binding of C4BP core protein to CD40 can be observed by precipitation of the C4BP core protein by sCD40:Ig but not by murine IgG2a. Furthermore, the core protein will bind sCD40:Ig over a similar concentration range as the full-length C4BP protein .

Example 3 - Proliferation of peripheral blood mononuclear cells (PBMCs) and of B cells in response to the addition of the C4BP core protein.

PBMCs or B cells (300, 000/well) are cultured in 96-well plates with the full-length C4BP protein as a positive control (at concentrations of 4, 20 or 100 micrograms/ml ) , or with the C4BP core protein (at concentrations of 0.1, 1, 5, 10 and 100 micrograms/ml) in RPMI plus 10% fetal calf serum (FCS) . As a negative control, cells are cultured in RPMI1640 plus 10% FCS alone. Proliferation is assayed after 4 days by measuring 3H- thymidine incorporation as described Jabara et al . , 1991, Journal of Immunology, volume 147, pages 1557 to 1560. The 3H- incorporation results show that proliferation of cells occurs with both the full-length and the core C4BP proteins, but not with the culture medium alone.

Example 4 - Injection of the C4BP core protein into mice.

This procedure may to used to observe the effects C4BP in vivo, and to confirm that it mimics the effect of an anti-CD40 agonist monoclonal antibody, by causing a rise in the plasma concentrations of the cytokines interleukin 12 and of interferon gamma.

The assay is carried out essentially as described by Turner et al., 2001, Journal of Immunology, volume 166, pages 89-94. As a positive control, 0.5 milligrams of the agonist anti-CD40 antibody FGK45.5 is injected intraperitoneally (ip) into 5-8 week old C57BL/6 mice. As a negative control, 0.5 milligrams of BSA is injected ip into control mice. In the experimental groups of mice, either 5 micrograms, 50 micrograms or 100 micrograms of the C4BP core protein is injected. Twenty-four hours after the injections, the mice are sacrificed, and serum are prepared from each mouse. These sera are then assayed for both interleukin 12 and interferon gamma using a commercially available murine cytokine assay (purchased from Genzyme, Cambridge MA). The results show that both the FGK45.5 monoclonal antibody and the C4BP core protein but not BSA cause significant increases in the plasma concentrations of both of the cytokines .

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described invention will be apparent to those of skill in the art without departing from the scope and spirit 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.

Claims

CLAIMS :

1. A method of agonising CD40 on the surface of a cell, which method comprises bringing the cell into contact with an effective amount of a C4BP core protein.

2. The method of claim 1, wherein said C4BP core protein is of SEQ ID NO:l or a fragment comprising residues 6-52 of SEQ ID N0:1.

3. A method of treating a subject which method comprises administering to the subject an effective amount of a C4BP core protein or vector encoding said protein.

4. The method of claim 3 wherein said subject has a disease associated with a defect in CD40L activity or expression.

5. The method of claim 3 or 4 wherein said subject has a condition characterised by a chronic autoimmune inflammatory process .

6. The method of claim 5 wherein said chronic autoimmune inflammatory process is rheumatoid arthritis.

7. The method of claim 3 or 4 wherein the subject has a condition selected from the group consisting of human X-linked hyper-IgM syndrome, combined variable immune deficiency (CVID) and post congenital rubella.

8. The method of claim 3 wherein said C4BP core is administered to a subject with a condition which will benefit from an enhanced B-cell response.

9. The method of claim 8 wherein said subject has a CD40- negative tumour.

10. The method of claim 8 wherein the subject has a B-cell lymphoma .

11. The method of claim 8 wherein the subject has a breast tumour .

12. The method of claim 8 wherein the C4BP is administered in conjunction with a bacterial, viral or parasitic antigen.

13. The method of claim 12 wherein the antigen is an antigen present in Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitides .

14. The method of claim 3 wherein said C4BP core is administered to a subject with a condition which will benefit from an enhanced T-cell response.