WO2009127666A2 - Method and compositions - Google Patents

Abstract

Vaccine compositions and methods for their use in the stimulation of immune responses in mammals, including humans, are provided. Also provided are methods for the prevention and treatment of cancer. These relate to breaking immune tolerance to selfantigens, inducing CD4+ and CD8+ T-cell responses, and inducing antibody responses in subjects without recourse to complex prime-boost schedules.

Description

Method and Compositions

FIELD OF THE INVENTION

This invention relates to vaccine compositions and their use in the stimulation of immune responses in mammals, especially humans, and in particular for the prevention and treatment of cancer.

BACKGROUND Inactivated whole organisms have been used in successful vaccination since the late nineteenth century. In more recent times, vaccines involving the administration of extracts, subunits, toxoids and capsular polysaccharides have been employed. Since genetic engineering techniques have been available, the use of recombinant proteins has been a favoured strategy, obviating many of the risks associated with use of purified proteins from natural sources.

Early vaccine approaches were based on the administration of proteins which stimulated some aspect of the immune response in vivo. Subsequently it was appreciated that immune responses could also be raised by administration of DNA which could be transcribed and translated by the host into an immunogenic protein.

The mammalian immune response has two key components: the humoral response and the cell-mediated response. The humoral response involves the generation of circulating antibodies which will bind to the antigen to which they are specific, thereby neutralising the antigen and eliciting its subsequent clearance by a process involving other cells that are either cytotoxic or phagocytic. B-cells are responsible for generating antibodies (plasma B cells), as well as holding immunological humoral memory (memory B-cells), i.e. the ability to recognise an antigen some years after first exposure to it, e.g., through vaccination. The cell mediated response involves the interplay of numerous different types of cells, among which are the T cells. T-cells are divided into a number of different subsets, mainly the CD4+ and CD8+ T cells.

Antigen-presenting cells (APC) such as macrophages and dendritic cells act as sentinels of the immune system, screening the body for foreign antigens. When extracellular foreign antigens are detected by APC, these antigens are phagocytosed (engulfed) inside the APC where they will be processed into smaller peptides. These peptides are subsequently presented on major histocompatibility complex class Il (MHC II) molecules at the surface of the APC where they can be recognised by antigen- specific T lymphocytes expressing the CD4 surface molecules (CD4+ T cells). When CD4+ T cells recognise the antigen to which they are specific on MHC Il molecules in the presence of additional adequate co-stimulatory signals, they become activated and secrete an array of cytokines that subsequently activate the other arms of the immune system.

In general, CD4+T cells are classified into T helper 1 (Th1 ) or T helper 2 (Th2) subsets depending on the type of response they generate following antigen recognition. Upon recognition of a peptide-MHC Il complex, TM CD4+ T cells secrete interleukins and cytokines such as interferon gamma thereby activating macrophages to release toxic chemicals such as nitric oxide and reactive oxygen/nitrogen species. IL-2 and TNF- alpha are also commonly categorized as Th1 cytokines. In contrast, Th2 CD4+ T cells generally secrete interleukins such as IL-4, IL-5 or IL-13.

Other functions of the T helper CD4+ T cells include providing help to activate B cells to produce and release antibodies. They can also participate to the activation of antigen- specific CD8+ T cells, the other major T cell subset beside CD4+ T cells.

CD8+ T cells recognize the peptide to which they are specific when it is presented on the surface of a host cell by major histocompatibility class I (MHC I) molecules in the presence of appropriate co-stimulatory signals. In order to be presented on MHC I molecules, a foreign antigen needs to directly access the inside of the cell (the cytosol or nucleus) such as is the case when a virus or intracellular bacteria directly penetrate a host cell or after DNA vaccination. Inside the cell, the antigen is processed into small peptides that are redirected to the surface of the MHC I molecules. Upon activation CD8+ T cells secrete an array of cytokines, such as interferon gamma that activates macrophages and other cells. In particular, a subset of these CD8+ T cells secretes lytic and cytotoxic molecules (e.g. granzyme, perforin) upon activation. Such CD8+ T cells are referred to as cytotoxic T cells. More recently, an alternative pathway of antigen presentation involving the loading of extracellular antigens or fragments thereof onto MHC I complexes has been described and called "cross-presentation".

Cells including cancer/tumour cells express many hundreds even thousands of genes. In the early 1980s, Van Pel and Boon published the discovery of cytolytic T cells directed against an antigen presented on tumour cells. This led to the characterization of the first tumour-specific, shared antigen: Melanoma Antigen GEne (MAGE-1 ; also named MAGE-A1 )(see, e.g., accession no. NP_004979). It was followed by the identification of a large number of genes sharing same expression pattern: The MAGE-1 gene belongs to a family of 12 closely related genes, MAGE 1 (above), MAGE 2 (see, e.g., accession no. P43356), MAGE 3 (see, e.g., accession no. NP_005353), MAGE 4 (see, e.g., accession no. NP_001011550), MAGE 5 (see, e.g., accession no. P43359), MAGE 6 (see, e.g., accession no. BAA06842), MAGE 7 (see, e.g., nucleic acid accession no. U10692), MAGE 8 (see, e.g., accession no. P43361 ), MAGE 9 (see, e.g., accession no. P43362), MAGE 10 (see, e.g., accession no. P43363), MAGE 11 (see, e.g., accession no. P43364), MAGE 12 (see, e.g., accession no. P43365), located on chromosome X and sharing with each other 64 to 85% homology in their coding sequence (De Plaen, 1994). These are sometimes known as MAGE A1 , MAGE A2, MAGE A3, MAGE A4, MAGE A5, MAGE A6, MAGE A7, MAGE A8, MAGE A9, MAGE A 10, MAGE A11 , MAGE A 12 (the MAGE A family). Two other groups of proteins are also part of the MAGE family although more distantly related. These are the MAGE B and MAGE C group. The MAGE B family includes MAGE B1 (also known as MAGE Xp1 , and DAM 10)(see, e.g., accession no. P43366), MAGE B2 (also known as MAGE Xp2 and DAM 6)(see, e.g., accession no. 015479), MAGE B3 (see, e.g., accession no. 015480) and MAGE B4 (see, e.g., accession no. 015481 ); the Mage C family currently includes MAGE C1 (see, e.g., accession no. O60732) and MAGE C2 (see, e.g., accession no. Q9UBF1 ).

MAGE antigens are expressed in a wide range of tumour types such as melanoma; breast cancer; liver cancer; bladder cancer including transitional cell carcinoma; lung cancer including non-small cell lung cancer (NSCLC); head and neck cancer including squamous cell carcinoma and oesophagus carcinoma; colon carcinoma; seminoma; and multiple myeloma. MAGE antigens are not expressed in normal cells, except testis and the placenta. However, this should not lead to antigen expression to the immune system as these germ line cells do not express MHC class I molecules. From their peculiar expression profile, the name of Cancer Testis (CT) genes was proposed for these genes. A new generation of cancer treatments based on antigens such as cancer testis antigens; antigens over-expressed in tumour tissue compared to normal tissue such as Her-2/neu (see, e.g., accession no. NP_004439.2); and tissue restricted tumour antigens such as P501 (also known as prostein) (see, e.g., accession no. NP_149093) are being developed. The strategy behind many of these therapies, often referred to as antigen specific cancer immunotherapy (ASCI), is to stimulate the patient's immune system into fighting the cancer. These therapies may be advantageous because side effects of taking such treatments may be minimal in comparison to side effects encountered by patients undergoing traditional non-targeted cancer treatment, such as chemotherapy.

Cancer testis antigens may represent promising targets for immunotherapy because generally they are expressed only by tumour tissue immune-privileged normal tissue such as testis and placenta, and some immune response has been detected in cancer patients to these antigens, even though through the action of a number of complexed mechanisms the body ultimately fails to fight the cancer.

Antigens that are highly over-expressed in cancer tissue may also be very useful targets for antigen-specific cancer immunotherapy, as well as tissue-specific antigens. For example, the Her-2/neu antigen is over-expressed in about 20 to 30% of all breast cancers (also referred to a c-erb B2 gene amplication); the Wilm's tumour suppression gene (WT1 ) (see, e.g., accession no. CAC39220) is thought to be over-expressed in certain leukaemias, kidney cancer and the like.

Tissue-specific antigens that may be useful for ASCI include the cancer prostate antigen P501 (also known as prostein). This antigen is over-expressed in prostate cancer and, although it is also expressed in normal prostate tissue, the normal prostate may be removed before commencing treatment using the ASCI.

The immune system has mechanisms to protect against development of immune responses to self-antigens: these mechanisms are known as immune tolerance. In order to stimulate the immune system to recognise tumour tissue, mechanisms of tolerance must be overcome. In certain protein based cancer immunotherapeutics this "tolerance" has been addressed by using potent adjuvants, such as those comprising MPL & QS21 and a TLR 9 agonist, for example CpG. The nature of the T-cell response is also influenced by the composition of the adjuvant used in a vaccine. For instance, adjuvants containing MPL & QS21 have been shown to activate Th1 CD4+ T cells to secrete IFN-gamma (Stewart et al. Vaccine. 2006, 24 (42-43):6483-92).

Whereas adjuvants are well known to have value in enhancing immune responses to protein antigens, they have not generally been used in conjunction with DNA-based vector vaccination. There are several hypotheses as to why adjuvants have not been used in conjunction with DNA-vector based vaccines. Indeed, interferences between the adjuvant and the vector may have an impact on their stability. In addition, one might expect that adding an adjuvant to an attenuated vector could increase the reactogenicity induced by the vector. If the body overreacts to the vector the immune system can become swamped and the desired effect to the antigen "lost".

On the other hand, there has been a report of enhancement of the efficacy of an adjuvanted DNA-based vector vaccine (Ganne et al. Vaccine (1994) 12(13) 1 190-1 196). In particular, the enhanced efficacy of a replication-defective adenovirus-vectored vaccine by the addition of oil adjuvants was correlated with higher antibody levels but the impact on CD4 and CD8 T cell responses was not reported.

It is generally thought that stimulation of both CD4+ and CD8+ cells are needed for optimal protective immunity, especially for the treatment of cancer.

SUMMARY OF THE INVENTION

Compositions, combinations, or a kit of parts are provided herein, said compositions, combinations, or a kit of parts comprising:

(a) a tumour associated antigen or derivative thereof, and

(b) a viral vector encoding the antigen or derivative.

Typically the viral vector of component (b) encodes the antigen or derivative of component (a).

Also provided are compositions, combinations, or a kit of parts comprising: (a) a first tumour associated antigen or derivative thereof, and (b) a viral vector encoding a second tumour associated antigen or derivative thereof in which the first antigen or derivative thereof differs from the second antigen or derivative thereof but in which the first and second antigen or derivatives thereof comprise one or more of the same epitopes or comprise one or more epitopes from the same tumour associated antigen.

By "epitope" is meant a portion of an immunogen or antigen to which the T-cell receptor responds (T cell epitope) or a site on an immunogen or antigen against which an antibody will be produced and to which it will bind (B cell epitope). In one embodiment of the present invention, the epitopes are immunodominant epitopes from the same tumour associated antigen. In an alternative embodiment, common epitopes are provided by a fusion partner protein.

By "viral vector" is meant any viral vector that is unable to replicate in vivo.

In one embodiment of the present invention, the composition or combination as described herein may also comprise an immunostimulant comprising a saponin or saponin derivative (such as Quil A or QS21 ); a TLR 4 ligand (such as monophosphoryl lipid A or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) ), and a TLR 9 agonist, for example a CpG containing oligonucleotide.

The compositions or combinations or kits of the invention may allow the immune system to recognise and initiate a response to the tumor associated antigen. Furthermore because the elements are given concomitantly, it is believed that a broader spectrum immune response may be generated, i.e., both a CD4+ and/or CD8+ T cell response and/or an antibody response to the antigen may be initiated.

As used herein the term "concomitantly" means components (a) and (b) are administered within a period of no more than 12 hours; or within a period of no more than 1 hour; or on one occasion, e.g. in the course of a visit to a health professional. For example, components (a) and (b) may be administered sequentially (as separate injections) within 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes or less than these times. Alternatively, components (a) and (b) may be administered simultaneously (for example, as separate injections or as a single injection). Although components (a) and (b) will typically be administered by injection, other routes of administration are contemplated and are within the scope of the invention.

Also provided are methods of eliciting or enhancing an immune response to a tumour antigen in a mammal, which comprise administering to a subject a composition or combination as described herein. Methods of inhibiting the development of a cancer and/or treating cancer in a mammal comprising administering to a subject a composition or combination as described herein are also provided.

Also provided are uses of a composition or combination as described herein in the manufacture of a medicament for eliciting or enhancing an immune response to a tumour antigen. Further provided are uses of a composition or combination as described herein in the manufacture of a medicament for inhibiting the development of a cancer (i.e. prevention of cancer) and/or treating cancer in a mammal.

In principle, based on our results in animal models, we believe that the methods described herein may be most useful in the prevention of cancer. Nevertheless use in the treatment of cancer is contemplated as an aspect of the invention.

In one embodiment of the present invention the mammal is a human patient.

The terms "protein," "antigen," and "immunogenic polypeptide" are used interchangeably in the context of the present specification.

Constructs of the present invention may include N-terminal or C-terminal His residues (including His residues immediately following an initial Met). For example, typically up to 6 His residues may be employed to facilitate isolation of the protein.

Constructs which have significant sequence identity, e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the whole length of a native sequence may be employed. Such constructs may produce a protein that has a similar function and enable an immune response to be generated to the native sequence. In some embodiments, up to 20, e.g., up to 10, e.g., 1 -5 susbtitutions (e.g., conservative substitutions) may be tolerated. Nucleic acids which differ from the native sequences, but which encode native proteins, or proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the whole length of the native amino acid sequence may be employed.

Sequence identity may be determined by conventional means, e.g., using the Basic Local Alignment Search Tool (BLAST), which identifies regions of local similarity between sequences.

The components of the vaccine of the invention may be co-formulated or co- administered. In one embodiment component (a) may be co-formulated with an immunostimulatory adjuvant and this co-formulated component (a) may be coadministered with component (b). In another embodiment both components (a) and (b) are co-formulated.

By "co-formulated" is meant that the elements are contained within the same composition, e.g., a pharmaceutical composition and/or is contained within the same vial or tube for a single administration.

In general a merit of the present invention is that useful immune responses, especially CD8 responses, are raised without recourse to complex prime-boost schedule. The convenience is greatest when the components of the formulation are co-formulated (so called "one-pot").

In one embodiment the tumor associated antigen or derivative thereof encoded by the viral vector (component b) is the same as the polypeptide antigen (component a) in the composition of combination according to the invention.

Suitably the antigens of component (a) and component (b) share one or more identical amino acid sequences of 5 or 6 amino acids; of 10 amino acids or more; of 15 amino acids or more; and/or of 25 amino acids or more. In one embodiment the antigens of components (a) and (b) may have an overall sequence identity of more than 90% over a stretch of 20 amino acids or more, e.g., 40 amino acids or more, e.g., 60 amino acids or more. In one embodiment the tumour associated antigen or derivative as described herein comprises at least one T cell epitope. In a further embodiment the tumour associated antigen or derivative as described herein comprises at least one B cell epitope.

Maintenance therapy may be required following initial treatment. This may comprise further vaccinations with the composition or combination according to the invention. Maintenance therapy may, for example be given at 1 , 2, 3, 4, 5 or 6 monthly intervals for 1 , 2, 3, 4, 5 or more years. This forms a further aspect of the invention.

SUMMARY OF THE SEQUENCE LISTINGS

SEQ ID NO: 1 - Amino acids 1 -127 of H influenzae Protein D

SEQ ID NO: 2 - An exemplary Protein D-MAGE fusion protein

SEQ ID NO: 3 - LAGE-I a amino acid sequence

SEQ ID NO: 4 - LAGE-I b amino acid sequence SEQ ID NO: 5 - NY-ESO-1 amino acid sequence

SEQ ID NO: 6 - human Her-2/neu amino acid sequence

SUMMARY OF THE FIGURES

Figure 1 : Amino acids 1 -127 of H influenzae Protein D (SEQ ID NO: 1 ) (amino acids 20-

127 are underlined)

Figure 2: An exemplary Protein D-MAGE fusion protein (SEQ ID NO: 2) in which the underlined portion = signal sequence of Protein D including 1 -Met and the substitutions

2-Asp and 3-Pro for the native aa 2-Lys and 3-Thr of protein D; the double underlined portion = amino acids 20 to 127 of Protein D; MD. = unrelated amino acids Met-Asp at aa 128-129 to create a cloning site; GG = unrelated amino acids GIy-GIy at 442-443;

Dotted.. underlined = fragment of MAGE3; amino acids 3-314 of MAGE3 (312 amino acids in total); and HHHHHHH = 7 His tail

Figure 3:Results post injection 1 in the analysis of ICS on PBL (see Example 3) Figure 4: Results post injection 2 in the analysis of ICS on PBL (see Example 3)

Figures 5-10: Results for Groups 1 , 3, 4, 5, 6, 8 respectively in the chromium release assay (see Example 3).

Figure 1 1 : Results for tumour growth (see Example 3).

Figures 12-14: Frequency of CD8 T cells producing cytokines in Balb/C mice after 1 , 2 and 3 injections respectively (see Example 4). Figure 15: Results for tumour growth (see Example 4).

DETAILED DESCRIPTION Antigens

Tumour antigens suitable for use in the present invention include proteins expressed in prostate cancer, breast cancer, colorectal cancers, lung cancer, kidney cancer, ovarian cancer, liver cancer and head and neck cancer, among others.

Cancer testis antigens that may be used in the present invention include the MAGE A family of antigens MAGE-A1 , A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12; also known as MAGE-1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12), the MAGE B antigens MAGE B1 , B2, B3 and B4, the MAGE C antigens MAGE-C1 and MAGE-C2 , the LAGE 1 antigen, the LAGE 2 antigen (also known as NY-ESO-1 ) and the GAGE antigen (see, e.g., accession no. NP_001465). The complete LAGE-I a amino acid sequence is set forth in the sequence listing as SEQ ID NO:3. The complete LAGE-I b amino acid sequence is set forth in the sequence listing as SEQ ID NO:4. The complete NY-ESO-1 amino acid sequence is set forth in the sequence listing as SEQ ID NO:5. General information relating to these proteins is available from the LICR web site (see ^w^M_Λgg_.Qggli^m _.. ^m _..y_.DJJ_.y-θ^|"g/Ctdatabase).

Prostate specific antigens may also be used in the present invention. Examples of prostate specific antigens that may be used include six-transmembrane epithelial antigen of the prostate (STEAP) (see, e.g., accession no. Q9UHE8), Prostate Specific Antigen (PSA) (see, e.g., AAA60192), prostatic acid phosphatase (PAP) (see, e.g., accession no. AAB60640), prostate stem cell antigen (PSCA) (see, e.g., accession no. AAC39607), prostate-specific membrane antigen (PSMA) (see, e.g., accession no. AAA60209) or the antigen known as prostase (see, e.g., AAF81227) (also known as P703P).

In one embodiment, the prostate antigen is P501 S or a fragment thereof. P501 S, also named prostein, is a 553 amino acid protein. Immunogenic fragments and portions of

P501 S comprising at least 20, 50, or 100 contiguous amino acids, or fragments comprising between 20-50 or 50-100 contiguous amino acids, may be used as the tumour associated antigen or derivative of the present invention. In one embodiment the tumour associated antigen or derivative is the PS108 antigen (disclosed in WO98/50567) or prostate cancer-associated protein (see WO99/67384). In some embodiments, fragments are amino acids 51 -553, 34-553 or 55-553 of the full-length P501 S protein. These can be expressed in yeast systems, for example DNA sequences encoding such polypeptides can be expressed in yeast systems.

In one embodiment, the antigen may comprise or consist of WT-1 expressed by the Wilm's tumor gene, or its N-terminal fragment WT-1 F comprising about or approximately amino acids 1 -249.

In one embodiment of the invention the tumour associated antigen or derivative is a breast cancer antigen, for exampleHer-2/neu, mammaglobin (see, e.g., accession no. AAC39608) or a B305D antigen.

The Her-2/neu antigen for use in the present invention may comprises the entire extracellular domain (ECD; for example the sequence comprising approximately amino acids 1-645 or 1 -653 of the amino acid sequence of Her-2/neu) or fragments thereof. Alternatively or additionally the construct may comprise at least an immunogenic portion of or the entire intracellular domain: for example approximately the C terminal 580 amino acids of the Her-2/neu sequence. See SEQ ID NO: 6.

One construct that may be used as the tumour associated antigen derivative of the present invention is a fusion protein of the ECD and the phosphorylation domain (PD) of Her-2/neu (ECD-PD). A further construct that may be used is a fusion protein of the ECD and a fragment of the phosphorylation domain of Her-2/neu (ECD-ΔPD). The Her- 2/neu fusion proteins and constructs as described may be derived from human, rat, mouse or simian/monkey Her-2/neu. Exemplary sequences and constructs of Her- 2/neu are described in WO00/44899.

PRAME (also known as DAGE) (see, e.g., the sequence of U.S. Patent Nos. 5,830,753 and 6,339,149) is another antigen that may be used as the tumour associated antigen of the present invention. Fusion proteins as described herein that comprise the PRAME antigen may also be used.

Colorectal antigens may also be used as the tumour associated antigens of the present invention. Examples of colorectal antigens that could be used include: C1585P (MMP 11 ) (see, e.g., accession no. NP_005931 ) and C1491 (E1A Enhancer Binding Protein) (see, e.g., accession no. NP_001073143 ), CASB618 (as described in WO00/53748); CASB7439 (as described in WO01/62778); and C1584 (Cripto) (see, e.g., the sequences of Cripto-1 and Cripto-3 in U.S. Patent Nos. 5,256,643; 5,654,140; 5,264,557; 5,620,866; and 5,650,285).

The invention also extends to use of the above antigens, immunogenic derivatives and immunogenic fragments and fusion proteins comprising same in aspects of the present invention.

Derivatives, fragments and fusion proteins

Tumour associated antigens of the present invention may be employed in the form of derivatives or fragments thereof rather than the naturally-occurring antigen.

As used herein the term "derivative" refers to an antigen that is modified relative to its naturally occurring form. The derivative may include a mutation, for example a point mutation. In one example, the derivative may change the properties of the protein, for example by improving expression in prokaryotic systems or by removing undesirable activity,, e.g., enzymatic activity. Derivatives of the present invention are sufficiently similar to native antigens to retain antigenic properties and remain capable of allowing an immune response to be raised against the native antigen. Whether or not a given derivative raises such an immune response may be measured by a suitably immunological assay such as an ELISA or flow cytometry.

In one embodiment of the present invention the derivative of the tumour associated antigen of the present invention is a fusion protein comprising a tumour associated antigen linked to a heterologous fusion partner protein. By "heterologous" with respect to a tumour associated antigen is intended a protein or polypeptide sequence that would not be linked to the tumour associated antigen in nature, i.e., is linked to the tumour associated antigen by deliberate human intervention.

The antigen and heterologous fusion partner protein may be chemically conjugated or may be expressed as recombinant fusion proteins. In one embodiment, a fusion protein of the present invention may allow increased levels of the fusion protein to be produced in an expression system compared to non-fused protein. Thus the fusion partner protein may assist in providing T helper epitopes, for example T helper epitopes recognised by humans (ie. The fusion partner protein is acting as an immunological fusion partner). The fusion partner may assist in expressing the protein at higher yields than the native recombinant protein (i.e., the fusion partner protein acting as an expression enhancer). In one embodiment, the fusion partner protein may act as both an immunological fusion partner and expression enhancing partner.

Fusion partner proteins may, for example, be derived from protein D. Protein D is a lipoprotein (a 42 kDa immunoglobulin D binding protein exposed on the surface of the Gram-negative bacterium Haemophilus influenzae). The protein is synthesized as a precursor with an 18 amino acid residue signal sequence, containing a consensus sequence for bacterial lipoprotein (see WO 91/18926). Native precursor Protein D protein is processed during secretion and the signal sequence is cleaved. The Cys of the processed Protein D (at position 19 in the precursor molecule) becomes the N terminal residue of the processed protein and is concomitantly modified by covalent attachment of both ester-linked and amide-linked fatty acids. The fatty acids linked to the amino-terminal Cysteine residue then function as membrane anchor.

In one embodiment, the tumour associated antigen derivative for use in the present invention may comprise Protein D or a derivative thereof as a fusion partner protein.

The protein D or a derivative thereof as described herein may comprise, for example: the first or N-terminal third of processed protein D or approximately or about the first or N-terminal third of processed protein D. In one embodiment, the protein D or a derivative thereof may comprise the first or N-terminal 100 to 1 15 amino acids of processed protein D; or the first or N-terminal 109 amino acids of processed protein D. In one embodiment, the native processed Protein D amino acids 2-Lys and 2-Leu may be substituted with amino acids 2-Asp and 3-Pro.

In one embodiment, the protein D or derivative thereof may further include the 18 or 19 amino acid signal sequence of precursor protein D. In one embodiment, the fusion partner protein derived from protein D comprises or consists of amino acids 20 to 127 of precursor protein D. In one embodiment of the present invention, the two amino acids 21 -Lys and 22-Leu of the precursor protein D fusion partner protein may be substituted with amino acids 21 -Asp and 22-Pro. The protein D fusion partner protein as described herein may additionally or alternatively contain deletions, substitutions or insertions within the amino acid sequence when compared to the wild-type precursor or processed protein D sequence. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more amino acids may be inserted, substituted or deleted. The amino acids may be substituted with conservative substitutions as defined herein, or other amino acids may be used.

In one embodiment, the fusion partner protein may comprise or consist of the protein shown in SEQ ID NO: 1 . In one embodiment, the fusion partner protein may comprise or consist of the amino acids underlined in Figure 1 , i.e., amino acid residues 20 through 127 of SEQ ID NO: 1 . An exemplary Protein D-MAGE fusion is shown in SEQ ID No: 2.

In another embodiment of the present invention, fusion partner proteins may be selected from NS1 or LytA or derivatives thereof as described below.

NS1 (hemaggluttinin)

NS1 is a non-structural protein from the influenzae virus. In one embodiment, the tumour associated antigen derivative of the present invention may comprise NS1 or a derivative thereof as a fusion partner protein. The NS1 or derivative thereof may comprise the N terminal 1 to 81 amino acids thereof.

LytA

LytA is derived from Streptococcus pneumoniae. The C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. In one embodiment, the tumour associated antigen derivative of the present invention may comprise LytA or a derivative thereof as a fusion partner protein. The LytA or derivative thereof may comprise the repeat portion of the LytA molecule found in the C terminal end starting at residue 178. In one embodiment, the LytA or derivative thereof comprises residues 188 - 305 of C-LytA.

Immunogenic polypeptides for use in the present invention will typically be recombinant proteins produced, e.g., by expression in a heterologous host such as a bacterial host, in yeast or in cultured mammalian cells. The term "tumor associated antigen derivative" means a polypeptide which partially or wholly contains sequences which occur naturally in a tumor associated antigen or which bears a high degree of sequence identity thereto (e.g., more than 95% identity over a stretch of at least 10, e.g., at least 20 amino acids). Derivatives also include sequences having conservative substituitions. Conservative substitutions are well known and are generally set up as the default scoring matrices in sequence alignment computer programs.

In general terms, substitution within the following groups are conservative substitutions, but substitutions between the following groups are considered non -con served. The groups are:

i) Aspartate/asparagine/glutamate/glutamine ii) Serine/threonine iii) Lysine/arginine iv) Phenylalanine/tyrosine/tryptophane v) Leucine/isoleucine/valine/methionine vi) Glycine/alanine

Derivatives of the present invention may also include chemically treated sequences such as treatment with an aldehyde (such as formaldehyde or glutaraldehyde), carboxymethylation, carboxyamidation, acetylation and other routine chemical treatments. Constructs of the present invention having dehvatised free thiol residues may also be used in the present invention. In particular carboxyamidated or carboxymethylated thiol derivatives may be used.

In one embodiment of the present invention the tumor associated antigen derivative may be a MAGE antigen as described herein having derivatised free thiol residues. The derivatised free thiol residues may be a carboxyamide or carboxymethylated derivatives.

The tumour associated antigen derivative of the present invention may alternatively comprise a construct comprising more than one tumour associated antigen. In one embodiment of the present invention, the tumour associated antigen derivative may comprise two or more tumour associated antigens. The term "fragment" as used herein refers to fragments of a tumour associated antigen or derivative of the antigen which contain at least one epitope, for example a CTL epitope, typically a peptide of at least 8 amino acids. Fragments of at least 8, for example 8-10 amino acids or up to 20, 50, 60, 70, 100, 150 or 200 amino acids in length are considered to fall within the scope of the invention as long as the fragment demonstrates antigenicity, that is to say that the major epitopes (e.g., CTL epitopes) are retained by the fragment and the fragment is capable of inducing an immune response that cross-reacts with the naturally occurring tumour associated antigen. Exemplary fragments may be 8-10, 10-20, 20-50, 50-60, 60-70, 70-100, 100-150, 150-200 amino acid residues in length (inclusive of any value within these ranges). Further exemplary fragments comprise at least 8 but no more than 10, at least 10 but no more than 20, at least 20 but no more than 50, at least 50 but no more than 60, at least 60 but no more than 70, at least 70 but no more than 100, at least 100 but no more than 150, at least 150 but no more then 200, or at least 200 but no more than 300 amino acids. Further exemplary fragments comprise at least 8, 10, 20, 50, 60, 70, 100, 150 or 200 amino acids.

Adenovirus In one embodiment of the present invention, the viral vector of component (b) to be used is an adenoviral vector.

Adenoviruses (also referred to as "Ad" or "Adv") have a characteristic morphology with an icosohedral capsid consisting of three major proteins, hexon (II), penton base (III) and a knobbed fibre (IV), along with a number of other minor proteins, Vl, VIII, IX, Ilia and Iva2. The virus genome is a linear, double-stranded DNA with a terminal protein attached covalently to the 5' termini, which have inverted terminal repeats (ITRs). The virus DNA is intimately associated with the highly basic protein VII and a small peptide termed mu. Another protein, V, is packaged with this DNA-protein complex and provides a structural link to the capsid via protein Vl. The virus also contains a virus- encoded protease, which is necessary for processing of some of the structural proteins to produce mature infectious virus.

Over 100 distinct serotypes of adenovirus have been isolated which infect various mammalian species. 51 of these serotypes are of human origin. In one embodiment of the present invention, the adenoviral vector of component (b) may be derived from a human adenovirus.

Examples of human-derived adenoviruses that may be used in the present invention are Ad 1 , Ad2, Ad4, Ad5, Ad6, Ad 1 1 , Ad 24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50. In one embodiment, Ad5, Ad11 and/or Ad35 may be used.

The human serotypes of adenoviruses have been categorised into six subgenera (A-G) based on a number of biological, chemical, immunological and structural criteria.

Although Ad5-based vectors have been used extensively in a number of gene therapy trials, there may be limitations on the use of Ad5 and other group C adenoviral vectors due to pre-existing immunity in the general population due to natural infection with human adenoviruses. This is because Ad5 and other group C members tend to be among the most seroprevalent serotypes and immunity to existing vectors may develop as a result of exposure to the vector during treatment. These types of pre-existing or developed immunity to seroprevalent vectors may limit the effectiveness of gene therapy or vaccination efforts. Alternative adenovirus serotypes may thus constitute important targets in the pursuit of gene delivery systems capable of evading the host immune response.

One such area of alternative serotypes are those derived from non human primates, especially chimpanzee adenoviruses: it has been shown that chimpanzee ("Pan" or "C") adenoviral vectors induce strong immune responses to transgene products as efficiently as human adenoviral vectors. In one embodiment of the present invention the adenoviral vector for use in the present invention may be may be derived from a non- human mammalian host. In one embodiment of the present invention, the vector may be a chimpanzee or pan adenoviral vector.

Non-human primate adenoviruses can be isolated from the mesenteric lymph nodes of chimpanzees. Chimpanzee adenoviruses are sufficiently similar to human adenovirus subtype C to allow replication of E1 deleted virus in HEK 293 cells. Yet chimpanzee adenoviruses are phylogenetically distinct from the more common human serotypes (Ad2 and Ad5). Pan 6 is less closely related to and is serologically distinct from Pans 5, 7 and 9.

Thus one or more of the adenoviral vectors for use as the vector of the present invention may be derived from a non-human primate adenovirus, e.g., a chimpanzee adenovirus. In one embodiment, the vector for use in the present invention is selected from serotypes Pan5, Pan6, Pan7 and Pan9.

Adenoviral vectors for use in the invention may also be derived from more than one adenovirus serotype, and each serotype may be from the same or different source. For example they may be derived from more than one human serotype and/or more than one non-human primate serotype and they may be a chimaeric adenoviral vector. Methods for constructing chimaeric adenoviral vectors are disclosed in WO2005/001 103.

One example of adenoviruses of use in the present invention are adenoviruses which are distinct from prevalent naturally occurring serotypes in the human population such as Ad2 and Ad5. This may avoid induction of immune responses against the vector which may limit the efficacy of subsequent administrations of the same serotype by blocking vector uptake through, eg. Neutralising antibodies.

Thus, the adenovirus may be an adenovirus which is not a prevalent naturally occurring human virus serotype. Adenoviruses isolated from animals have immunologically distinct capsid, hexon, penton and fibre components but are phylogenetically closely related. Specifically, the virus may be a non-human adenovirus, such as a simian adenovirus and in particular a chimpanzee adenovirus such as Pan 5, 6, 7 or 9. Examples of such strains are described in WO03/000283 and are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 201 10-2209, and other sources. In one embodiment of the present invention, chimpanzee adenovirus strains that may be used include Pan 5 [ATCC VR-591], Pan 6 [ATCC VR-592], and Pan 7 [ATCC VR-593].

Use of chimpanzee adenoviruses may be advantageous over use of human adenovirus serotypes because of the lack of pre-existing immunity, in particular the lack of cross- neutralising antibodies, to adenoviruses in the target population. Cross-reaction of the chimpanzee adenoviruses with pre-existing neutralizing antibody responses is only present in 2% of the target population compared with 35% in the case of certain candidate human adenovirus vectors. The chimpanzee adenoviruses are distinct from the more common human subtypes Ad2 and Ad5, but are more closely related to human Ad4 of subgroup E, which is not a prevalent subtype. Pan 6 is less closely related to Pan 5, 7 and 9.

The adenovirus of the invention should generally be replication defective. This means that it has a reduced, and generally absent, ability to replicate in non-complementing cells, compared to the wild type virus. This may be brought about by mutating the virus e.g. by deleting a gene involved in replication, for example deletion of the E1 a, E1 b, E3 or E4 gene or part thereof (or more than one of these genes or part thereof).

The adenoviral vectors in accordance with the present invention may be derived from replication defective adenovirus comprising a functional E1 deletion. Thus the adenoviral vectors according to the invention may be replication defective due to the absence of the ability to express adenoviral E1 a and E1 b, i.e., are functionally deleted in E1 a and E1 b. The recombinant adenoviruses may also bear functional deletions in other genes [see WO03/000283] for example, deletions in E3 or E4 genes. The adenovirus delayed early gene E3 may be eliminated from the adenovirus sequence which forms part of the recombinant virus. The function of E3 is not necessary to the production of the recombinant adenovirus particle. Thus, it is unnecessary to replace the function of this gene product in order to package a recombinant adenovirus useful in the invention. In one particular embodiment the recombinant adenoviruses have functionally deleted E1 and E3 genes. The construction of such vectors is described in Roy et al., Human Gene Therapy 15:519-530, 2004. Chimpanzee adenoviral vectors with various early gene deletions are described in WO03/46124. Recombinant adenoviruses may also be constructed having a functional deletion of the E4 gene, although it may be desirable to retain the E4 ORF6 function. Adenovirus vectors according to the invention may also contain a deletion in the delayed early gene E2a. Deletions may also be made in any of the late genes L1 through to L5 of the adenovirus genome. Similarly deletions in the intermediate genes IX and Iva may be useful.

Other deletions may be made in the other structural or non-structural adenovirus genes. The above deletions may be used individually, i.e., an adenovirus sequence for use in the present invention may contain deletions of E1 only. Alternatively, deletions of entire genes or portions thereof effective to destroy their biological activity may be used in any combination. For example in one exemplary vector, the adenovirus sequences may have deletions of the E1 genes and the E4 gene, or of the E1 , E2a and E3 genes, or of the E1 and E3 genes (such as functional deletions in E1 a and E1 b, and a deletion of at least part of E3), or of the E1 , E2a and E4 genes, with or without deletion of E3 and so on. Such deletions may be partial or full deletions of these genes and may be used in combination with other mutations, such as temperature sensitive mutations, to achieve a desired result.

The adenoviral vectors can be produced in any suitable cell line in which the virus is capable of replication. In particular, complementing cell lines which provide the factors missing from the viral vector that result in its impaired replication characteristics (such as E1 and/or E4) can be used. Without limitation, such a cell line may be HeLa, A549, HEK 293, KB, Detroit [e.g., Detroit 510] and WI-38 cells, among others. Other suitable parent cell lines may be obtained from other sources, such as PER.C6 cells or Her 96 cells (Crucell).

Alternative viral vectors can be derived from adeno-associated viral vectors (AAVs), measles, lentiviruses, alphaviruses, bacloviruses, herpes simplex virus, and poxviruses such as cowpox, fowlpox (avipox), pigeonpox, canarypox, suipox and sheeppox/goatpox. The polynucleotide sequences which encode immunogenic polypeptides may be codon optimised for mammalian cells. Such codon-optimisation is described in detail in WO05/025614.

In one embodiment the viral vector contains a spacer sequence of about 15 base pairs as, for example described in WO97/21826.

In one embodiment of the present invention the polynucleotide constructs comprise an N-terminal leader sequence. The signal sequence, transmembrane domain and cytoplasmic domain are individually all optionally present or deleted. In one embodiment of the present invention all these regions are present but modified.

A promoter for use in the adenoviral vector according to the invention may be the promoter from HCMV, i.e., gene, for example wherein the 5' untranslated region of the HCMV, i.e., gene comprising exon 1 is included and intron A is completely or partially excluded as described in WO02/36792.

Maintenance therapy as described herein may, for example, be specifically designed to use a different viral vector or different serotype to that used in the original/initial treatment.

When several antigens are fused into a fusion protein, such protein may be encoded by a polynucleotide under the control of a single promoter.

In an alternative embodiment of the invention, several antigens may be expressed separately through individual promoters, each of said promoters may be the same or different. In yet another embodiment of the invention some of the antigens may form a fusion, linked to a first promoter and other antigen(s) may be linked to a second promoter, which may be the same or different from the first promoter.

Thus, the adenoviral vector may comprise one or more expression cassettes each of which encode one antigen under the control of one promoter. Alternatively or additionally it may comprise one or more expression cassettes each of which encode more than one antigen under the control of one promoter, which antigens are thereby expressed as a fusion. Each expression cassette may be present in more than one locus in the adenoviral vector.

The polynucleotide or polynucleotides encoding immunogenic polypeptides to be expressed may be inserted into any of the adenovirus deleted regions, for example into the E1 deleted region.

Although two or more polynucleotides encoding immunogenic polypeptides may be linked as a fusion, the resulting protein may be expressed as a fusion protein, or it may be expressed as separate protein products, or it may be expressed as a fusion protein and then subsequently broken down into smaller subunits.

Adjuvant

Adjuvants are described in general in Vaccine Design - the Subunit and Adjuvant Approach, e.g., Powell and Newman, Plenum Press, New York, 1995.

Suitable adjuvants include an aluminium salt such as aluminium hydroxide or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.

In the formulation of the invention it may be preferred that the adjuvant composition preferentially induces a Th1 response. However it will be understood that other responses, including other humoral responses, are not excluded.

It is known that certain vaccine adjuvants are particularly suited to the stimulation of either TM or Th2 - type cytokine responses. Traditionally the best indicators of the Th1 :Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Th1 or Th2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the lgG1 :lgG2a ratio of antigen specific antibody responses.

Thus, a Th1 -type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Th1 -type cytokines in vivo (as measured in the serum) or ex vivo (cytokines that are measured when the cells are re-stimulated with antigen in vitro), and induces antigen specific immunoglobulin responses associated with TM -type isotype.

Preferred Th1 -type immunostimulants which may be formulated to produce adjuvants suitable for use in the present invention include and are not restricted to the following: The Toll like receptor (TLR) 4 ligands, especially an agonist such as a lipid A derivative particularly monophosphoryl lipid A or more particularly 3 Deacylated monophoshoryl lipid A (3D - MPL; GlaxoSmithKline).

3 D -MPL primarily promotes CD4+ T cell responses characterized by the production of IFN-Y (Th1 cells, i.e., CD4 T helper cells with a type-1 phenotype). It can be produced according to the methods disclosed in GB 2 220 21 1 A. Chemically it is a mixture of 3- deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably in the compositions of the present invention small particle 3D- MPL is used. Small particle 3D -MPL has a particle size such that it may be sterile-filtered through a 0.22μm filter. Such preparations are described in International Patent Application No. WO94/21292.

Synthetic derivatives of lipid A are known and thought to be TLR 4 agonists including, but not limited to: OM174 (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o- phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D- glucopyranosyldihydrogenphosphate), (see WO95/14026)

OM 294 DP (3S, 9 R) -3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-

[(R)-3-hydroxytetradecanoylamino]decan-1 ,10-diol,1 ,10-bis(dihydrogenophosphate) (see WO99 /64301 and WO 00/0462 )

OM 197 MP-Ac DP (3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-

9-[(R)-3-hydroxytetradecanoylamino]decan-1 ,10-diol,1 -dihydrogenophosphate 10-(6- aminohexanoate) (see WO01/46127)

Other TLR4 ligands which may be used are Alkyl Glucosaminide Phosphates (AGPs) such as those disclosed in WO9850399 or US6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in US6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants. Saponins are also preferred Th1 immunostimulants in accordance with the invention. Saponins are well known adjuvants. For example, Quil A (derived from the bark of the South American tree Quillaja Saponaha Molina), and fractions thereof, are described in US5,057,540 and EP 0 362 279 B1 . The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No. 5,057,540 and EP 0 362 279 B1 . Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil-A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al. (1991 . J. Immunology vol 146, 431 -437). Combinations of QS21 and polysorbate or cyclodexthn are also known (see WO99/10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO96/33739 and WO 96/1 171 1 . One such system is known as an lscom and may contain one or more saponins.

The adjuvant of the present invention may in particular comprise a Toll like receptor (TLR) 4 ligand, for example 3D-MPL, in combination with a saponin.

Other suitable adjuvants include TLR 9 ligands (agonists). Thus another preferred immunostimulant is an immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (see WO96/02555, EP 468520, Davis et al., J.Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.Immunol., 1998, 161 (9):4463-6). Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in immunostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimulatory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the CG motif is not methylated, but other unmethylated CpG sequences are known to be immunostimulatory and may be used in the present invention. In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequences containing oligonucleotides can activate various immune subsets, including natural killer cells (which produce interferon γ and have cytolytic activity) and macrophages (see Wooldrige et al VoI 89 (no. 8), 1977). Other unmethylated CpG containing sequences not having this consensus sequence have also now been shown to be immunomodulatory.

CpG when formulated into vaccines, is generally administered in free solution together with free antigen (see WO96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (see WO98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al. Supra ; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

Other TLR9 agonists of potential interest include immunostimulatory CpR motif containing oligonucleotides and YpG motif containing oligonucleotides (Idera).

Such immunostimulants as described above may be formulated together with carriers, such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide). For example, 3D-MPL may be formulated with aluminium hydroxide (EPO 689 454) or oil in water emulsions (see WO95/17210); QS21 may be advantageously formulated with cholesterol containing liposomes (see WO96/33739), oil in water emulsions (see WO95/17210) or alum (see WO98/15287); CpG may be formulated with alum (Davis et al. Supra ; Brazolot-Millan supra) or with other cationic carriers. Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (see WO94/00153; WO95/17210; WO96/33739; WO98/56414; WO99/12565; WO99/11241 ), more particularly the combination of QS21 and 3D-MPL as disclosed in WO94/00153. Alternatively, a combination of CpG plus a saponin such as QS21 may form a potent adjuvant for use in the present invention.

In one embodiment, the saponin may be formulated in a liposome or in an ISCOM. In one embodiment, the saponin component as described herein may be combined with an immunostimulatory oligonucleotide.

Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt (e.g., as described in WO00/23105). One system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in WO 96/33739. This combination may additionally comprise an immunostimulatory oligonucleotide.

Thus one example of an adjuvant for use in the present invention comprises QS21 and/or MPL and/or CpG.

In an alternative embodiment there is provided an adjuvant formulation comprising QS21 , 3D-MPL & tocopherol in an oil in water emulsion (see WO 95/17210).

Another formulation that may be used in the present invention comprises a CpG oligonucleotide alone or together with an aluminium salt.

Thus, adjuvants that may be used in the formulations according to the invention are as follows: i) 3D-MPL + QS21 in a liposome (see, e.g., Adjuvant B in the example section below) ii) Alum + 3D-MPL iii) Alum + QS21 in a liposome + 3D-MPL iv) Alum + CpG v) 3D-MPL + QS21 + oil in water emulsion vi) CpG vii) 3D-MPL + QS21 (e.g., in a liposome or oil in water) + CpG viii) QS21 + CpG.

In one embodiment, the adjuvant is presented in the form of a liposome, ISCOM or an oil-in-water emulsion. In one example embodiment of the invention the adjuvant comprises an oil-in-water emulsion. In another example embodiment of the invention the adjuvant comprises liposomes. Suitably the adjuvant component does not contain any virus or virus derived component other than the viral vector of component (b) of the composition or combination.

In a further aspect of the present invention there is provided a method of manufacture of a vaccine formulation as herein described, wherein the method comprises admixing one or more first immunogenic polypeptides according to the invention with a suitable adjuvant.

Compositions, dosage and administration

In methods of the invention, the immunogenic polypeptide(s), the adenoviral vector(s) and the adjuvant are administered concomitantly.

Typically the adjuvant will be co-formulated with an immunogenic polypeptide. Suitably the adjuvant will also be co-formulated with any other immunogenic polypeptide to be administered. Typically the adenoviral vector is contained in a composition, e.g., a pharmaceutical composition. Alternatively, the one or more first immunogenic polypeptides, the one or more adenoviral vectors and an adjuvant are co-formulated.

Thus, there are provided compositions according to the invention which comprise one or more immunogenic polypeptides, one or more adenoviral vectors, and an adjuvant.

Compositions and methods according to the invention may involve use of more than one immunogenic polypeptide and/or more than one adenoviral vector. Use of multiple antigens is especially advantageous in raising protective immune responses to certain cancers. Compositions according to the invention may comprise more than one adjuvant.

Compositions and methods employed according to the invention may typically comprise a carrier, e.g., an aqueous buffered carrier. Protective components such as sugars may be included. Compositions should be administered in sufficient amounts to transduce the target cells and to provide sufficient levels of gene transfer and expression and to permit cancer-specific immune responses to develop thereby to provide a prophylactic or therapeutic benefit without undue adverse or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the liver, inhalation, intranasal, intravenous, intramuscular, intratracheal, subcutaneous, intradermal, infusion, epidermal, rectal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the gene product or the condition. The route of administration primarily will depend on the nature of the condition being treated. Most suitably the route is intramuscular, intradermal or epidermal.

When the first immunogenic polypeptide, adjuvant and adenoviral vector are not co- formulated, the different formulations (e.g., polypeptide/adjuvant and adenoviral vector formulations) may be administered by the same route of administration or by different routes of administration.

Therapies according to the invention may be combined with other cancer therapies, for example immunotherapies such as those based on cancer testis antigens, chemotherapy, radiotherapy and/or surgery.

Dosages of compositions in the methods will depend primarily on factors such as the condition being treated, the age, weight and health of the subject, and may thus vary among subjects. For example, a therapeutically effective adult human dosage is generally in the range of from about 100 μl_ to about 100 ml_ of a carrier containing concentrations of from about 1 x 10⁶ to about 1 x 10¹⁵ particles, about 1 x 10¹¹ to 1 x 10¹³ particles, or about 1 x 10⁹ to 1 x 10¹² particles of virus together with around 1 - 1000ug, or about 2-100ug, e.g., around 4-40ug immunogenic polypeptide. Dosages will range depending upon the individual and the route of administration. For example, a suitable human for intramuscular injection is in the range of about 1 x 10⁹ to about 5 x 10¹² virus particles and 4-40 ug protein per ml_, for a single site. One of skill in the art may adjust these doses, depending on the route of administration, and the therapeutic or vaccinal application for which the composition is employed.

The amount of adjuvant will depend on the nature of the adjuvant and the immunogenic polypeptide, the condition being treated and the age, weight and health of the subject. Typically for human administration an amount of adjuvant of 1 -100ug, e.g., 10-50 ug per dose may be suitable. Suitably an adequate immune response is achieved by a single concomitant administration of the composition or compositions of the invention in methods of the invention.

Adjuvant preparations

1) The preparation of oil in water emulsion followed the protocol as set forth in WO 95/17210.

The emulsion contains: 42.72 mg/ml squalene, 47.44 mg/ml tocopherol, 19.4 mg/ml Tween 80. The resulting oil droplets have a size of approximately 180 nm Tween 80 was dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml two fold concentrate, emulsion 5g of DL alpha tocopherol and 5ml of squalene were vortexed until mixed thoroughly. 90ml of PBS/Tween solution was added and mixed thoroughly. The resulting emulsion was then passed through a syringe and finally microfluidised by using an M1 10S microfluidics machine. The resulting oil droplets have a size of approximately 180 nm

2) Preparation of oil in water emulsion with QS21 and MPL

Sterile bulk emulsion was added to PBS to reach a final concentration of 500 μl of emulsion per ml (v/v). 3D-MPL was then added. QS21 was then added Between each addition of component, the intermediate product was stirred for 5 minutes. Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8 +/- 0.1 with NaOH or HCI. The final concentration of 3D-MPL and QS21 was 100 μg per ml for each.

3) Preparation of liposomal MPL A mixture of lipid (such as phosphatidylcholine either from egg-yolk or synthetic) and cholesterol and 3 D-MPL in organic solvent, was dried down under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as phosphate buffered saline) was then added, and the vessel agitated until all the lipid was in suspension. This suspension was then microfluidised until the liposome size was reduced to about 100 nm, and then sterile filtered through a 0.2 μm filter. Extrusion or sonication could replace this step.

Typically the cholesterol: phosphatidylcholine ratio was 1 :4 (w/w), and the aqueous solution was added to give a final cholesterol concentration of 10 mg/ml. The final concentration of MPL is 2 mg/ml. The liposomes have a size of approximately 100 nm and are referred to as SUV (for small unilamelar vesicles). The liposomes by themselves are stable over time and have no fusogenic capacity.

4) Preparation of Adjuvant B

Sterile bulk of SUV was added to PBS. PBS composition was Na₂HPO₄: 9 mM; KH₂PO₄: 48 mM; NaCI: 100 mM pH 6.1 . QS21 in aqueous solution was added to the SUV. The final concentration of 3D-MPL and QS21 was 100 μg per ml for each. This mixture is referred as Adjuvant B. Between each addition of component, the intermediate product was stirred for 5 minutes. The pH was checked and adjusted if necessary to 6.1 +/- 0.1 with NaOH or HCI.

Embodiments are specifically envisaged where aspects of the invention comprising a certain element or elements are limited to said aspects consisting or consisting essentially of the relevant elements as separate embodiments.

The below non-limiting examples are shown to illustrate exemplary methodology of the present invention.

EXAMPLES

Example 1 - WT1 antigen

A truncated WT1 recombinant protein has been developed, deleted from the Zn Finger portion. When injected 2 to 4 times in HLA-A2 Tg mice, this protein formulated in adjuvant induced a high titer of antibodies, and CD4 and CD8 T cells which produce IFNγ. In CB6F1 or C57BL/6 mice the protein induced antibodies and (CD4).

GSK has demonstrated that co-injection of an adjuvanted recombinant protein with a recombinant adenovirus coding for the same protein further improved the CD4 and CD8 responses.

Aim:

Experiments to be performed - evaluate the impact of a prime- boost approach (adjvuanted protein - adenoviral vector) on the immune response and anti tumor efficacy (therapeutic and prophylactic settings) induced by adjuvanted WT1 protein.

- evaluate the immune response and anti-tumor efficacy (therapeutic and prophylactic settings ) when both adjuvanted WT1 protein and adenovirus are coadministered.

Experiment 1 : Therapeutic model

CB6F1 mice are implanted with WT1 -expressing tumor cell line on day 0.

On days 7, 14 and 21 mice receive immunizations according to the study plan below.

Group Immunization at 7 day intervals

(n = 12 in each group; WT1 protein is adjuvanted)

Immune responses to WT1 are evaluated by harvesting splenocytes 2- 3 weeks after the final immunization and testing for the production of cytokines by CD4+ and CD8+ cells by FACS assay. Additionally, mouse sera are tested for the antibody response to WT-1 . Tumor size are measured until day 28 or 35.

Experiment 2: Protection model

CB6F1 mice are vaccinated in the same groups as shown below. In order to gain statistical power in the analysis of the results, there are 20 mice per group. 12 mice per group are sacrificed at day 42 and their splenocytes harvested for analysis of the immune response. Immune analyses are performed as described in experiment 1 (intracellular cytokine staining and FACS analysis and antibody response). In the remaining 8 mice per group, on day 42 WT1 - expressing tumor cells are implanted sub-cutaneously. Tumor growth is monitored for up to 4 - 5 weeks.

Group Immunization at 14 day intervals

(n = 20 in each group; WT1 protein is adjuvanted)

Example 2 - HER2 Project Proposal:

Background: A truncated dHER2 recombinant protein has been developed comprising the ECD domain fused to the phosphorylation region of the ICD domain. When injected in mice, this protein formulated in a GSKBio adjuvant induced a high titer of antibodies and T cells (CD4 -CD8) which produce IFNy (by ICS). The immune response induced a long term protection in mice against challenge with a murine cell line engineered to express HER2 (TC1 HER2).

GSK demonstrated that co-injection of an adjuvanted recombinant protein with a recombinant adenovirus coding for the same protein further improved the CD4 and CD8 Responses.

Experiments to be performed

- evaluate the impact of a prime- boost approach (dHER2 adjvuanted protein - adenoviral vector Ad5-HER2) on the immune response and anti tumor efficacy (therapeutic and prophylactic settings) induced by the adjuvanted HER2 protein.

- evaluate the immune response and anti-tumor efficacy (therapeutic and prophylactic settings when both the adjuvanted dHER2 protein and the adenovirus are co-administered. Experiment 3: Therapeutic model

CB6F1 mice are implanted with the murine TC1 cell line expressing human HER2 on day O. On days 7 and 14 mice receive immunizations according to the study plan below, with either HER2 protein, adenovirus, or a combination.

Group Immunization at 7 day intervals

n = 12/gp; dHER2 protein is adjuvanted

Immune responses to HER2 are evaluated by harvesting splenocytes 1 -2 weeks after the final injection and testing for cytokine production in a FACS assay. Additionally, mouse sera are tested for the antibody response to HER2. Tumor size is measured until day 28.

Experiment 4: Protection model

CB6F1 mice are vaccinated in the same groups as shown below. In order to gain statistical power in the analysis of the results there are 20 mice per group. 12 mice per group are sacrificed at day 28 and their splenocytes harvested for analysis of the immune response. Immune analysis is performed as described in experiment 3.

In the remaining 8 mice per group, on day 28 TC1 tumor cells are implanted sub- cutaneously. Tumor growth is monitored for up to 4 weeks.

Group Immunization at 14 day intervals

dHer2 protein is adjuvanted

Example 3 - MAGE A3 antigen Aim: to evaluate the immune response obtained when Chimpadeno MAGE-A3 was injected before, after, or concomitantly with MAGE-A3 adjuvanted protein and to see the impact on tumour growth in a therapeutic setting.

Materials: All experiments were performed in CB6/F1 mice.

MAGE A3 - The protein D-MAGE A3 fusion protein having the sequence of SEQ ID

NO: 2 was used (see eg WO99/40188 for methods of preparation)

Adjuvant X is based on 3D-MPL and QS21 in liposomes (see e.g. Adjuvant B) with added CpG. The dose of MAGE A3/Adjuvant X injected into mice contained 5ug 3D-MPL, 5ug QS21 and 42ug CpG and 1 ug of MAGE A3 protein.

Chimpadeno MAGE-A3 = An E1 deleted Pan7 adenovirus containing MAGE-A3 as transgene.

Methods:

The experiment was composed of 8 groups of 9 CB6F1 mice that received at day 0 TC1 MAGE-A3 tumour cells (10e5/mouse) followed 7 days later by 2 injections at one week interval ( day 7 and 14) of either o Group 1 : PBS o Group 2: MAGE-A3 (1 μg) o Group 3: Chimpadeno MAGE-A3 ( 10e10 viral particles) o Group 4: Adjuvant X o Group 5: MAGE-A3/Adjuvant X o Group 6: MAGE-A3/Adjuvant X at day 7 + Chimpadeno MAGE-A3 at day 14 o Group 7: Chimpadeno MAGE-A3 at day 7 + MAGE-A3/Adjuvant X at day 14 o Group 8: MAGE-A3/Adjuvant X/Chimpadeno MAGE-A3 at days 7 and 14 (1 vial) The following read-outs were performed:

- ICS on PBL (post first and second injection)

- Chromium release assay (post second injection)

- Measurement of the tumour growth twice/week

Results:

ICS on PBL:

The frequency of CD4 and CD8 T cell producing cytokines (IFNg and/or TNFa) is measured by flow cytometry (FACS) after 2 hours in vitro restimulation with a pool of 15 mer peptides overlapping by 1 1 , covering the entire MAGE-A3 sequence, followed by overnight incubation with brefeldin.

Results of ICS on PBL (IFNg/TNFa production = average pf 3 pools of 3 mice per group) are shown in Figure 3 (results after priming, 1 injection) and Figure 4 (results after booster, 2 injections).

After the priming injection, groups 6, 7 and 8 showed a CD8 response.

Co-injection of MAGE-A3 protein with adjuvant and Chimpadeno MAGE-A3 gave rise to slightly better CD8 response.

After the booster injection, all groups receiving both MAGE-A3 protein and Chimpadeno MAGE-A3 in prime boost setting show a better CD8 response than either MAGE-A3 protein with adjuvant or Chimpadeno MAGE-A3 alone. The second injection of MAGE- A3 protein with adjuvant and Chimpadeno MAGE-A3 increased the CD8 response relative to a single injection. The CD8 response following two injections of MAGE-A3 protein with adjuvant and Chimpadeno MAGE-A3 combined was superior to two injections of MAGE-A3 protein with adjuvant or two injections of Chimpadeno MAGE- A3, but not superior to an injection of MAGE-A3 protein with adjuvant followed by an injection of Chimpadeno MAGE-A3 or an injection of Chimpadeno followed by an injection of MAGE-A3 protein with adjuvant. Chromium release assay

In order to assess the lytic potential of CD8 responses induced, a chromium release assay has been performed on the spleens of the immunised mice after the booster injection.

The chromium release assay was performed on all groups (1 pool/group) except groups 2 and 6. The experiment was conducted 14 days after the second injection. 40.10e6 spleen cells were incubated with 1 ug/ml/CD8 immunodominant MAGE-A3 3 peptide ("pept 57") ina 6 well plate (10ml/plate). After 7 days, the effector T cell population was mixed with Cr51 targets - CT26 (this is a colon carcinoma from mice), CT26 MAGE-A3, CT26 pulsed with the CD8 dominant MAGE-A3 peptide ("pept 57"), CT26 pulsed with the irrelevant peptide TRP2, TC1 , TC1 MAGE-A3, TCI pulsed with the CD8 dominant MAGE-A3 peptide and TCI pulsed with the irrelevant peptide TRP2 - at different effecter/target ratios (ranging from 100:1 to 0.3:1 ) in a 96 well plate. After 4 hrs the supernatants were harvested by removing 100ul from each well manually. Chromium release was counted in a Y counter. Results were expressed in cpm or % of lysis

% specific lysis = (experimental release-spontaneous release) x100 (maximum release-spontaneous release) maximum release =cpm in wells containing Triton spontaneous release = cpm in control wells (with medium)

Results for groups 1 , 3, 4, 5, 7 and 8 are shown, respectively, in Figures 5-10. No specific CTL lysis was found when spleen cells were re-stimulated with the TRP2 peptide, as expected.

No specific CTL lysis was found on the TCI based targets.

Specific lysis (up to 55%) was found only on the target CT26 pulsed with teh specific

CD8 peptide for MAGE-A3 especially in the groups receiving Chimpadeno MAGE-A3 alone or the combination of both MAGE-A3 plus adjuvant and Chimpadeno MAGE-A3 given either as a prime boost or injected concomitantly.

Tumour growth:

Results are shown in Figure 11. No therapeutic effect on tumour growth was shown with MAGE-A3 protein or Chimpadeno MAGE-A3 alone (same tumour growth curve as PBS). A little benefit was shown with Adjuvant X alone or when both MAGE-A3 plus adjuvant and Chimpadeno MAGE-A3 administered, either in prime boost or concomitantly. The greatest benefit was shown with MAGE-A3 plus Adjuvant X without combination with Chimpadeno MAGE-A3. Differences between the groups may not be statistically significant.

Example 4 - MAGE A3 antigen Aim: to evaluate the immune response obtained when Chimpadeno MAGE-A3 was injected before, after, or concomitantly with MAGE-A3 adjuvanted protein and to see the impact on tumour growth in a therapeutic setting.

Materials: All experiments were performed in Bab/c mice.

MAGE A3 - The protein D-MAGE A3 fusion protein having the sequence of SEQ ID

NO: 2 was used (see eg WO99/40188 for methods of preparation)

Adjuvant X is based on 3D-MPL and QS21 in liposomes (see e.g. Adjuvant B) with added CpG. The dose of MAGE A3/Adjuvant X injected into mice contained 5ug 3D-MPL, 5ug QS21 and 42ug CpG and 1 ug of MAGE A3 protein..

Chimpadeno MAGE-A3 = An E1 deleted Pan7 adenovirus containing MAGE-A3 as transgene.

Methods:

The experiment was composed of 7 groups of 12 female Balb/c mice that received at day 0 CT26MAGE-A3 cl 12 cells (10e6/mouse, subcutaneous) followed by 3 injections at days 3, 10 and 17 of either o Group 1 : PBS (days 3, 10, 17) o Group 2: MAGE-A3 (1 ug) /adjuvant Adjuvant X (=P) (days 3, 10,

17) o Group 3: Chimpadeno MAGE-A3 (10e10 viral particles) (=A) (days

3, 10, 17) o Group 4: Adjuvant X (days 3, 10, 17) o Group 5: P (days 3, 10) then A (day 17) o Group 6: A (day 3) then AP(days 10, 17) o Group 7: Chimpadeno MAGE-A3 + MAGE-A3/Adjuvant X ("one- pot") (days 3, 10, 17)

The following read-outs were performed:

- ICS on PBL (7 days post first, second and third injections) (4 pools of 3 mice/group)

- Measurement of the tumour growth twice/week

Results

ICS on PBL

CD4 responses: none after 1 injection; after 2 injections detectable but still very low; after 3 injections still very low.

CD8 responses are shown in Figures 12-14 (responses after 1 , 2 and 3 injections respectively). In these tumour bearing mice there is a background in the PBS and adjuvant groups after priming. After 2 injections the CD8 response is very high in the groups receiving MAGE-A3/Adjuvant X and/or the Chimpadeno MAGE-A3. After 3 injections the CD8 response is even higher than post 2 injections in the groups that responded, especially in group 5.

Tumour growth

Results are shown in Figure 15.

There was no therapeutic effect for any of the treatments.

Abbreviations

ASCI antigen specific cancer immunotherapy

CTL cytotoxic T lymphocyte

ICS Intracellular cytokine staining PBL peripheral blood lymphocytes

PBS phosphate buffered saline

Further abbreviations used in the Figures:

AdjX = Adjuvant X M3/Adj X ASCI = MAGE A3+Adjuvant X

Chimpadeno M3 = Chimpadeno MAGE A3

Chimpadeno = Chimpadeno MAGE A3

Adeno = Chimpadeno MAGE A3 ASCI = MAGE A3+Adjuvant X

2IM = two intramuscular injections

3IM = three intramuscular injections d = day

GR = Group

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications referred to herein are incorporated by reference in their entirety.

Claims

1 . A composition or combination comprising:

(a) a tumour associated antigen or derivative thereof, and

(b) a viral vector encoding a tumour associated antigen or derivative thereof.

2. A composition or combination according to claim 1 in which the viral vector of component (b) encodes the tumour associated antigen or derivative of component (a).

3. A composition or combination according to claim 1 in which the viral vector of part (b) encodes a tumour associated antigen or derivative that differs from the tumour associated antigen or derivative of component (a), and in which the tumour associated antigens or derivatives of components (a) and (b) comprise one or more of the same epitopes or comprise one or more epitopes from the same tumour associated antigen.

4. A composition or combination according to claim 3 in which the epitopes are immunodominant epitopes from the same tumour associated antigen.

5. A composition or combination according to claim 3 in which the epitopes are immunodominant epitopes from the same fusion partner protein.

6. A composition or combination according to any preceding claim in which component (a) further comprises an adjuvant.

7. A composition or combination according to any preceding claim in which component (b) further comprises an adjuvant.

8. A composition or combination according to claim 6 or 7, in which the adjuvant comprises one or more of the following components: a saponin or saponin derivative, a TLR 4 ligand and a TLR 9 ligand.

9. A composition or combination according to any of claim 8, in which the TLR 9 ligand is an oligonucleotide containing a CpG motif.

10. A composition or combination according to claim 8 or 9, in which the saponin or saponin derivative is QS21 .

1 1 . A composition or combination according to any one of claims 8 to 10, in which the TLR4 ligand is monophosphoryl lipid A.

12. A composition or combination according to any one of claims 6 to 10 in which the adjuvant comprises QS21 and 3D-MPL.

13. A composition or combination according to any one of claims 6 to 10 in which the adjuvant comprises QS21 , 3D-MPL and CpG.

14. A composition or combination according to any one of claims 6 to 13 in which the adjuvant contains an oil-in-water emulsion.

15. A composition or combination according to any one of claims 6 to 14 in which the saponin is formulated to form ISCOMS or liposomes.

16. A composition or combination according to any preceding claim, in which the antigen is selected from P501 S, MAGE-A3, WT1 and Her2/neu.

17. A composition or combination according to any preceding claim, in which the viral vector is an adeno viral vector.

18. A composition or combination according to claim 17, in which the adenoviral vector is derived from a non-human primate virus serotype.

19. A composition or combination according to claim 18 in which the non-human primate adenoviral vector is selected from chimpanzee adenovirus serotypes Pan5, Pan6, Pan7 and Pan9.

20. A composition or combination according to claim 19 in which the adenoviral vector is derived from a human virus serotype.

21. A composition or combination according to claim 20 in which the human adenoviral vector is selected from human adenovirus serotypes Ad1 , Ad2, Ad4, Ad5, Ad6, Ad 11 , Ad 24, Ad34 and Ad35.

22. A composition according to any preceding claim, in which the components of the composition or combination are co-formulated.

23. A method of raising an immune response against a tumour associated antigen which comprises administering a composition or combination according to any preceding claim, in which the tumour associated antigen of part (a) and the viral vector of part (b) are administered concomitantly.

24. A method of treating a patient suffering from cancer which comprises administering a composition or combination according to any of claims 1 to 23, in which the tumour associated antigen of part (a) and the viral vector of part (b) are administered concomitantly.

25. A method of preventing a patient from suffering from cancer which comprises administering a composition or combination according to any of claims 1 to 23, in which the tumour associated antigen of part (a) and the viral vector of part (b) are administered concomitantly.

26. Use of a composition or combination according to any of claims 1 to 23 in the manufacture of a medicament for stimulating an immune response in a mammal.

27. Use of a composition or combination according to any of claims 1 to 23 in the manufacture of a medicament for treating a patient suffering from cancer.

28. Use of a composition or combination according to any of claims 1 to 23 in the manufacture of a medicament for preventing a patient from suffering from cancer.