AU2003262216A1 - Composition for inducing a tumor-specific immune response, method for the production and utilization of said composition in the treatment of neoplasia - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): IMMUNOGENEC BIOTECHNOLOGIE GMBH Invention Title: COMPOSITION FOR INDUCING A TUMOR-SPECIFIC IMMUNE RESPONSE, METHOD FOR THE PRODUCTION AND UTILIZATION OF SAID COMPOSITION IN THE TREATMENT OF NEOPLASIA The following statement is a full description of this invention, including the best method of performing it known to me/us: Composition for the induction of a tumour-specific immune response, methods for its production and use of the composition for the treatment of neoplasia The present invention relates to a composition comprising a phage or a functionally equivalent fragment thereof expressing, on its surface, at least one tumour antigen as fusion protein with a phage coat protein or a functionally equivalent derivative thereof and, optionally, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable diluent. The present invention further relates to a method for cloning and expressing tumour antigens on phages by which a specific immune response is induced. Moreover, the invention relates to a composition produced by means of said method which is meant for use as injection preparation for specific immune therapy of neoplasia in mammals, particularly in humans.

The immune system is the body's own defence system dealing with the fight against pathogens. In principle, there are two kinds of defence systems against infectious factors: the specific acquired (adaptive) immune response and the non-specific inherent immune response. In the following, the adaptive immune response, in particular, which is only triggered by an antigen is of importance. The special features of the adaptive immune response are adjustment, memory and specificity. They are the consequence of a clonal selection process triggered by antigen contact.

The immune system has a humoral and cellular organisation. With the humoral immune response there is, amongst others, a destruction of antibody-loaded tumour cells by natural killer cells (NK cells) triggered by the antibodies bound to the cell surface of tumours contacting the NK cells. This antibody-dependant, cell-mediated cytotoxicity (ADCC) is a very effective mechanism for the destruction of tumour cells.

See also RJ. Dearman et al. 1988 (Blood 72: 1985-91) and George AJ. et al. 1987 (J.

Immunol. 140: 1695-170). Within the frame of the cellular immune response, too, the immune system sees to the fighting of tumour cells: An activation of T-lymphocytes, however, presupposes, in most cases, the interaction with antigen-presenting cells (APCs). Therefor, the T-cell requires another co-stimulatory signal which is delivered by the antigen-presenting cells. The lack of these co-stimuli means that, as a consequence, the T-cells no longer react to an activation. Due to the lack of costimulating molecules cancer cells can escape an activation of the immune system Some tumour cells can be recognised and destroyed by the immune system. This function of the immune system becomes clear e.g. by the spontaneous remissions.of tumours observed at times. With the regression of tumours T-lymphocytes, amongst others, play a role, wherein the T-cell reaction against a tumour presupposes the recognition of so-called tumour-specific transplant antigens (so-called TSTA). It is, however, still not clear why some tumours trigger a spontaneous adaptive immune response and others do not. Burnet calls the ability of the immune system to find and destroy tumour cells as "immune surveillance" (Burnet FM. 1970, The concept of immunological surveillance, Prog. Exp. Tumor. Res. 13, 1-27). Due to the wide spread of cancer diseases it can be seen, however, that the immune surveillance is not particularly effective or that, apparently, the tumour cells have immunological escape mechanisms which enable them to escape the effect of the functioning immune system.

Tumour cells can have, e.g. only little immunogenic effect since they lack MHC molecules or co-stimulatory molecules. Another possibility to escape the immune system is that tumour cells expressing antigens detectable by the immune system lose said antigens due to a antibody-induced absorption into the cell or due to antigenic variation (immune selection). After all, tumours often produce substances such as e.g. TGF-P which directly suppress the immune response.

In the past few years a variety of tumour-associated antigens have been described most of which were identified by means of monoclonal antibodies in mice (Reisfeld and Sell, eds.: Monoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular and Cellular Biology, Vol. 27, Alan R. Liss, 1985, NY, 1-609). In spite of most antigens having proven to be oncofetal or differentiation antigens and their tumour specificity constituting a quantitative but not a qualitative feature since they also occur on normal cells to a small extent, some antigens are also sufficiently specific for neoplastic cells in comparison with normal cells so that they can potentially be used for the identification of tumour cells and for therapy.

Some of the best characterized tumour-specific antigens are the idiotype proteins expressed by neoplastic lymphocytes (Stevenson et al. 1977: Idiotypic determinants on the surface immunoglobulin of neoplastic lymphocytes: a therapeutic target. Fed.

Proc.; 36: 2268-71). In this context an idiotype is the total of antigenic epitopes (idiotopes) in the variable region of an antibody. Lymphome cells can be recognised selectively via said idiotopes. Thus, it is not surprising that with immunotherapeutic strategies in lymphome therapy idiotype vaccination plays a central role.

Animal experiments of various kinds have shown that immunisation with tumour antigens can lead to the defence against viable cancer cells administered subsequently. A possible approach for this is the use of cell vaccines consisting either of living, killed or genetically modified cancer cells. The effectiveness of cell vaccination is based on the induction of cytotoxic T-cells (CTLs) which are able to specifically destroy tumour cells. So far, however, it seems to be the case that the effectiveness of cell vaccination is not particularly high since not enough cytotoxic Tcells can be recruited for an effective tumour therapy to be achieved. Another approach for immunotherapy of cancer diseases is the immunisation with tumour antigens. In this context, the destruction of tumour cells is mainly mediated via an antibody-dependant cell-mediated cytotoxicity (ADCC). In this context, one of the main problems is the weak immunogenicity of the tumour antigens. The reason for that is that the tumour-associated antigens representing the targets for such an immune response are often present in normal cells, even if only in small amounts, and are, thus, recognised by the immune system as "self" against which there is normally no immune response. Thus, it is not certain whether cancer patients can be treated effectively with this approach. In contrast, the idiotype protein, for example, is a tumour-specific antigen which is produced by lymphoma cells exclusively. As could be shown in mice and clinical studies, a partly very effective immune response can be induced against idiotype proteins. The problem with idiotype vaccination is that the idiotype proteins have to be produced for each patient individually and that idiotpye proteins themselves are only little immunogenic. Idiotype proteins, therefore, have to be coupled to an immunogenic carrier molecule, e.g. KLH so that an antiidiotypic immune response can be induced (George AJ. et al.: Idiotypic vaccination as a treatment for a B cell lymphoma. J. Immunol. 1988, 141: 2168-74; Levy R. et al.: Therapy of lymphoma directed at idiotypes. J. Natl. Cancer Inst. Monogr. 1990; 1990:61-8). Vaccination with fragments of the idiotype proteins such as the CDR3peptide is by far less effective. (Campbell MJ. et al.: Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits. J. Immunol. 1987; 139: 2825-33).

Furthermore, it turned out that correct folding is crucial for the effectiveness of immunotherapy which is essentially mediated via anti-idiotypic antibodies. Until recently, idiotype proteins had to be produced by means of time- and cost-consuming hybridoma techniques. Today, however, it is possible to produce a patient-specific idiotypic vaccine by recombinant techniques e.g. in form of a single-strand fragment (Hawkins RE. et al.: Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines. Blood 1994; 83: 3279-88). Said single-strand fragments have meanwhile been successfully tested as DNA vaccines (Spellerberg MB et al.: NA vaccines against lymphoma: promotion of anti-itiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment J. Immunol. 1997: 159: 1885-92 and King CA. et al.: DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma., Nat. Med. 1998; 4: 1281-6). Single chain (sc) idiotypic vaccines could also be produced in tobacco plants and used for vaccination successfully. (McCormick AA et al.: Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc. Natl. Acad. Sci. USA 1999; 96: 703-8). With the growing importance of tumours with regard to their absolute frequency, which is mainly due to demographic reasons, there are many endeavours to treat the cancer effectively by appropriate measures but, at the same time to treat the patients with care.

Phages are particles similar to viruses which infect bacteria and can multiply in them.

At present, A-bacteriophages are frequently used for forming cDNA libraries. For that purpose A-ZAP® vectors have proved to be suitable, as described in the US-A 5,128, 256 (Huse, Sorge, Short). In this patent specification a method is disclosed wherein genes can be cloned with the high efficiency of the phage system and recombinant phagemid vectors BlueScript® vectors) can be produced via the step of in vivo excision without further subcloning processes.

Filamentous phages are oblong stretched phage particles with a length of 7 x 900 nm. They can infect Gram-negative bacteria and multiply here. They consist of an oblong capsid in helical order surrounding the single-stranded virus DNA. The virus coat consists of five phage proteins which consists of the major coat protein cpVll and the four minor coat proteins cplll, cpVl, cpVII and cplX. Since the coat proteins cplll and cpVlll are secreted in the periplasmatic space of Gram-negative bacteria to be assembled here as a component of the phage surface, they are an ideal target molecules for the fusion with idiotypes (cf. US-A 5,658,727 (Barbas, Kang, Lerner), granted 19 August 1997). At present, filamentous phages are widely used within "Phage Display" technology. "Phage Display" technology means the expression of peptides or proteins in fusion with phage coat proteins on the phage surface. Particularly preferred for that are the coat proteins Ill and VIII. The gVIII protein consisting of 50 amino acids is one of the main components of the phage coat and is present in approx. 2700 copies in the coat: The gill protein with its 406 amino acids is essentially bigger, it is, however, only present in 3-5 copies at the infectious end of the phage. The gill protein plays an important role .with the infection of bacteria. Further coat proteins occurring about as frequently as the gill protein on the phage coat are the gVI, gVII and gIX protein with the gVI protein serving as anchor of the gill protein and the gVII and gIX proteins being located at the other end of the phage.

At present, the "Phage Display" technology is widely used, in particular, when identifying peptide ligands, in epitope mapping and when selecting recombinant antibodies.

The technology may, however, also be used to produce immunogenes. Therefore, a partly good (protective) immune response can be induced against antigenic epitopes expressed on phages both with and without adjuvants. See studies by de la Cruz et al. 1988 (JBC 263, 4318-22), Greenwood et al. 1991 (JMB 220:821-27), Minenkova et al. 1993 (Gene 128:85-88), Meola et al. Immunol. 154, 3162-72), Bastien et al.

1997 (Virol. 234, 118-22). Moreover, DE 69124800 T2 describes genetically modified filamentous phages which, amongst others, express immunogenic oligopeptides of FMDV or of the malaria pathogen Plasmodium falciparum on their surface.. The described immunogenes have in common that small antigenic oligopeptides expressed by phages are used for vaccination. There, peptides are expressed either in fusion with the gill or gVIll protein. By immunisation with said phages a specific Tcell dependant humoral immune response can be induced with or without addition of an adjuvant against the antigenic peptides specifically cross-reacting with the original antigenes (see Willis et al., Gene 1993; 128, 97-83), wherein an immunogenic effect preferably was detected with "gVIII vaccines" but, at least, "gVIII vaccines" were more effective than "gill vaccines", possible because they exhibit a higher antigen density on their surface. There is no indication in the literature that bigger proteins (or polypeptides) expressed by phages are effective as immunogenes. On the contrary, a person skilled in the art would even advise not to express complex proteins on phages, since by doing so the load with antigens would be multiplied and, thus, the induction of an immune response would either not be possible at all or not be specific enough. By the expression of bigger proteins or domains on phages it was to be expected not knowing the results of the present invention that the biological behaviour could be changed in an undesirable way which makes the use of phages vaccines unsafe.

In the patent specification DE 3703702 Al a vaccine against melanoma is described which essentially comprises pure antigenic peptides or proteins belonging to the melanoma-associated p97 antigen. The peptides or proteins can also be expressed by recombinant viruses, including bacteriophages, by means of which vaccine formulations on the basis of inactivated viruses or on the basis of active viruses can be produced. DE 3703702 Al does, however, not contain any teaching with regard to a possible method for the production of recombinant bacteriophages comprising said peptides or proteins belonging to p97. Due to steric reasons, however, it is not possible to produce recombinant phages with immunogenic proteins being expressed on their surface. It is only possible if phages hybrids are produced. Thus, there is only the possibility of a conjugation to a carrier molecule giving immunogenicity.

Accordlingly, the general concept to present antigenic structures belonging to p97 as fusion proteins with a coat protein of viruses or bacteriophages is not mentioned.

Inducement of a specific immune therapy against a tumour presupposes the knowledge of a tumour antigen. With knowledge of such antigen the immune therapy can be induced artifically by addition of a specific antibody or by immunisation with a tumour antigen. Therapy with monoclonal antibodies has, however, the disadvantage that the biological half-life in the body is only shodrt and, therefore, long-term antitumour effects are not being observed. Furthermore, the risk of tumour escape mechanisms is high, since the immune response is only directed against an epitope.

The advantage of active immune therapy by immunisation with a tumour antigen, however, is the long-lasting immune response. The advantages of said active immunisation can be seen very clearly in the example of idiotype vaccination: Induction of a polyclonal, lymphoma-specific and long-lasting immune response. In hospital, however, idiotype vaccination has not been applied a lot, since idiotype vaccines have to be produced for the patients individually which, up to now, has only been possible in complex, time-consuming and expensive hybridoma techniques.

The production of idiotype vaccines as soluble sc-fragments, however, presents the.

difficulty, apart from the folding problem, to produce these in bigger amounts and to purify them for therapeutic use. It cannot be expected either that the expressed tumour antigens necessarily are immunogens. Accordingly, the technical problem underlying the present invention was to provide a method which makes an inexpensive and fast production of patient-specific tumour vaccines possible.

Preferably, these should also be good immunogens.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge H:\GabdeaK(eepASpec,41442.99.doc 08/04103 in the art, in Australia or in any other country.

This technical problem has been solved by providing the embodiments characterized in the claims.

Thus, the present invention relates to a composition comprising a phage or a functionally equivalent fragment thereof expressing at least one tumour-specific and/or tumour-associated antigen as fusion protein with a phage coat protein or a fragment or a functionally equivalent derivative thereof on its surface and, optionally, a pharmaceutically acceptable carrier and/or a dendritic cell and/or a vehicle and/or a pharmaceutically acceptable diluent.

Within the meaning of the present invention the term "functionally equivalent fragment" of a phage comprises fragments of phage particles produced by physical, chemical and/or biological treatment and which differ due to said treatment(s) morphologically and/or structurally and/or functionally from native phage particles, however, exhibit essentially the same immunological, preferably immune-stimulating properties as native phage particles. A "functionally equivalent fragment" of a phage comprises, for example, phage particles whose genome was destroyed by chemical treatment without changing the nucleo capsid and/or the coat, or phage particles whose genome and/or nucleo capsid and/or coat has been changed by e.g.

enzymatic, heat and/or ultra-sound treatment.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Within the meaning of the present invention the term "functionally equivalent derivative" of a phage coat protein comprises a phage coat protein exhibiting an Nterminal and/or C-terminal and/or internal deletion and/or exhibiting an insertion, in addition to the naturally-occurring amino acid sequence, or/and whose amino-acid sequence differs from the naturally-occurring amino-acid sequence by one or more substitutions, while at least the ability to interact with other molecules the viral genome or other viral coat proteins) necessary for the virion assembly is maintained.

Suitable "fragments" are known to the person skilled in the art.

Within the meaning of the present invention the term "tumour-specific" and "tumourassociated" relates to antigens which are components of cancer cells and can 7 \\Velb_filesf-omneSJuanita\Keeppatent41442-99DIV+doc 13/11/03 derive from the cytoplasm, the cell surface or the nucleus. Preferably, said antigens are expressed on the tumour cell surface as surface antigens, they can, however, also be detectable as multiplied intracellularly. A presentation of a tumour cell can e.g. also take place in the context of MHC class II molecules.

According to the invention "tumour-specific antigens" are polypeptides which are exclusively formed by tumour cells. These can be tumour-specific neo-antigens which e.g. were formed due to gene rearrangement, mutations or generally as a consequence of a transformation in malignant cells. A typical example of a tumourspecific antigen is the idiotype protein expressed by lymphoma cells. Furthermore, tumour-specific antigens can also be proteins formed by activation of suppressed genes if they only occur in or on tumour cells. "Tumour-associated antigens", however, are, according to definition, antigens which preferably occur in or on tumour cells but also are detectable on non-malignant cells. They can, for example, be detectable as physiologically-occurring differentiation antigens or as intermediate products of the ontogenesis of a cell type which disappear from normal cells with further differentiation. Some of said antigens can also be detected increasingly with 7a Wnmelb_files\home$JuanitlaKeep patentl41442-99.DIVdoc 13/11/03 specific pathological processes in non-tumour cells. Examples of said antigens are oncofetal antigens such as the carcinoembryonic antigen (CEA) in colon carcinomas, the "squamous cell carcinoma" antigen (SSC) in epithelial tumours, a,-feto protein in prim. liver cell carcinoma, isoferritin and fetal sulfoglyco protein in gastric and colon carcinoma, a2-H ferro protein in early-childhood malignoma or y-feto protein in sarkomas, leukaemia and mammary carcinoma. Other examples according to the invention are the Tennessee antigen (Tennagen), tissue-polypeptide antigen TPA, onkofetal membrane antigens (OFMA), tumour-specific transplantation antigens (TSTA), membrane-associated antigens (MATA) as well as crypt antigens such as the "A-like" antigen.

Furthermore, "tumour-associated antigens" within the meaning of the invention are polypeptides produced in low copy number on normal, non-tumourous cells and in high copy number by tumour cells so that, in this case, the specificity for the tumour cell is not qualitative but quantitative. Preferably, tumour-associated antigens are presented in a copy number enabling a quantitative determination e.g. via flowcytometric methods, wherein the signal intensity ("mean fluorescence" value) is the same as or higher than the signal intensity in normal cells, however preferrably at least higher by the factor 10 or one log-level higher than in normal cells, which can clearly be seen by a shift of the curve to the right in the single parameter histogram.

An example of said antigen is the onkogen Bcl-2 which is clearly overexpressed e.g.

in lymphoma cells. Furthermore, tumour-associated antigens within the meaning of the present invention are further antigens making an effective tumour therapy possible, even if said therapy is non-specific.

Within the meaning of the present invention the terms "tumour-specific antigen" and "tumour-associated antigen" also comprise fragments of the above-mentioned surface antigens essentially corresponding to the relevant extracellular domains.

Preferably, the surface antigens or fragments thereof have a length of at least amino acids, preferably 30, 40 or 50, more preferably a number of amino acids great enough for the length comprising at least one immunogenic domain of the tumour antigen in their three-dimensional structure and, most preferably, the complete primary amino-acid sequence of a native tumour antigen. Moreover, the abovementioned terms within the meaning of the present invention comprise surface antigens and fragments thereof whose amino-acid sequence deviates by one or more substitutions from the amino-acid sequence occurring naturally, while the, amino-acid substitutions preferably represent conservative exchanges. Such exchanges comprise, for example, the exchange of a neutral, hydrophobic amino acid, the exchange of a neutral, polaric one, the exchange of an alcaline one or the exchange of an acidic amino acid by an amino acid of the respective same class which are known to the person skilled in the art.

Preferably, the composition according to the invention is a pharmaceutical composition.

In a particularly preferred embodiment the pharmaceutical composition is a vaccine.

As mentioned above, the composition of the invention can comprise a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable diluent.

Examples of suitable carriers are known to the person skilled in the art and comprise, e.g. phosphate-buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents or detergents, sterile solutions etc.

Compositions comprising said carriers can be produced by means- of known conventional methods.

The compositions according to the invention can be administered to an individual in suitable dosage. Administration can take place orally or parenterally, e.g. in an intravenous, an intraperitoneal, a subcutan, perinodal, an intramuscular, a topic, an intradermal, intranasal or intrabronchial way or via a catheter at a place in the artery.

The amount of the dosage is determined by the attending doctor and essentially depends on clinical factors. These factors are known in medicine and science and comprise, e.g. the body size and the weight, the body surface, the age, the sex and the general physical condition of the patient, the specific composition to be administered, the length of treatment, the kind of administration and the possible simultaneous treatment with other pharmaceutical compositions. A typical dosage can be within a range e.g. between 0.001 and 1000 pg, while dosages can possible be below or above the range given as an example, especially under consideration of the above-mentioned factors. In general, the dosage should be within a range of 1 pg and 10 mg units per day when the composition of the invention is administered regularly. If the composition is administered intravenously, which is not preferably recommended to minimise the risk of an anaphylactic reaction, the dosage should be within a range of 1 pg and 10 mg units per kilogram body weight per minute.

The composition of the invention can be administered locally or systemically.

Compositions for a parenteral administration comprise sterile aqueous or nonaqueous diluents, suspensions and emulsions. Examples of non-aqueous solvents are propylenglycol, polyethylenglycol, vegetable oils such as olive oil, and organic ester compounds such as ethyloleate, which are suitable for injection. Aqueous carriers comprise water, alcoholic-aqueous solutions, emulsions, suspensions, saline solutions and buffered media. Parenteral carriers comprise sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate and bound oils.

Intravenuous carriers comprise e.g. fluid, nutrition and electrolyte supplements (as e.g. the ones based on Ringer's dextrose). The composition according to the invention can further comprise preservatives and other additives, such as e.g.

antimicrobial compounds, antioxidants, complex formers and inert gases.

Furthermore, depending on the intended use, compounds such as interleukines, growth factors, differentiation factors, interferons, chemotactic proteins or a nonspecific immunomodulating agent can be contained.

Due to the composition according to the invention, surprisingly, a tumour-specific immune response is triggered, which exerts a protective immunity among Vertebrata against an administration of tumour cells and is essentially based on a humoral but also on a cellular immune response. It could be shown in vitro that dendritic cells pulsed with the phage vaccines of the invention induce a T-cell proliferation. It could further be shown in cytotoxicity assays that a specific lysis of the lymphoma cells occurs due to Bcl-2 expressing phages. The Daudi-idiotypic phages carried along as controls, however, could induce no cytotoxic immune response, as expected, since Daudi lymphoma cells express no histocompatibility antigens due to the 132microglobulin defect and, thus, are not receptable for cytotoxic T-cell response.

These data suggest that via antigen-presenting cells a specific T-cell-depending immune response can be triggered in vitro. These data further suggest that said properties play an important role in vivo, too, and, thus, apart from the -expected humoral immune response, a specific cell-mediated anti-tumour response is induced, by which the effect of the phage vaccine is potentially enhanced. One of the essential features is likely to be the preferred absorption of the phages by antigen-presenting cells, by which an effective loading with antigens takes place. Contrary to general expectations the loading with antigens did not prove to be too high apparantly.

The composition of the vaccine of the invention allows for a fast and inexpensive production of tumour-specific phage vaccines, wherein, preferably, knowledge of immunogenic epitopes is not necessary. Equally, the ligands of the tumour antigens do not have to be characterized in detail either, nor do the expressed tumour antigens have to exhibit the same functional effects. A further advantage is the fact that an immunisation against several epitopes of a tumour antigen follows the use of complex proteins, which induces a polyclonal immune response directed against the tumour and, thus, reduces the risk of tumour escape mechanism. This effect can, within the meaning of the present invention, be further enhanced by the expression of several different tumour antigens on phages in the form of a polyvalent vaccine. A further advantage is that epitopes, due to the composition according to the invention, can be recognised not only in their linear form, as is the case when using oligopeptides, but also in their three-dimensional structure-dependant form. Said properties are particularly advantageous since they are the prerequisites e.g. for effective idiotypic vaccination. With the idiotypic vaccines it can further be seen that the presentation of the idiotypic proteins on phages has a very advantageous effect on immunisation, since the immune response against only weak immunogenic idiotypic proteins is enhanced by the phages and thereby only becomes fully usable.

The injection preparation according to the invention can, in principle, be used in any mammal and, particularly, also in humans to induce, in the fight against neoplasia, a polyclonal, antibody-mediated immune response directed against the tumour. The advantage of said compositions is a long-term protection of the individual. The composition is, as already mentioned above, e.g. in the form of a sterile solution, emulsion or suspension. It can optionally contain a suitable antimicrobial substance, tools for isotonication as well as isohydria or euhydria. Furthermore, substances for the chemical stabilisation antioxidants) and substances for the non-specific enhancement of the immune response, such as the aluminium hydroxide, can be added. The additives are to improve the immunising effect without influencing the tolerability in a negative way.

In a preferred embodiment the composition of the invention is an injection preparation for the specific immune therapy of neoplasia in mammals, particularly preferred in humans.

In a further preferred embodiment the composition according to the invention is .an injection preparation for the patient-specific treatment of lymphoma, particularly non- Hodgkin lymphoma or an injection preparation for the specific immune therapy of malignant melanoma, urogenital, gynecological and/or gastrointestinal carcinoma, thyroid carcinoma and/or bronchial carcinoma.

In ae most preferred embodiment the composition according to the invention is designed for the subcutaneous, intradermal, perinodal or intraperitoneal application/administration.

In another preferred embodiment of the composition according to the invention the phage or the functionally equivalent fragment thereof expresses at least one cytokine on its surface. Said cytokine, advantageously is an interleukin, preferably IL-1a, IL- 113, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15 and/or IL-18, a growth or differentiation factor, preferably GM-CSF, G-CSF, M-CSF and/or Flt3-Ligand, a member of the TNF-family, preferably TNF-a, LT-a, LT-p, OX-40, 4-1BB or CD40 ligand, a chemotactic protein, preferably MCP-1, an interferon, preferably IFN-a and/or IFN-y, or another immunomodulating agent. Cytokines can also be viral proteins with similar functional effect or fragments or analogous proteins which have the same functional effect as the cytokine.

The at least one cytokine is expressed preferably as fusion protein with a phage coat protein or a functionally equivalent derivative thereof.

In another preferred embodiment of the composition of the present invention the phage belongs to the family of the Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Lipothrixviridae, Microviridae, Myoviridae, Plasmaviridae, Podoviridae, Siphoviridae, Sulpholobus shibatae viruses (SSV) or Tectiviridae.

In a particularly preferred embodiment the phage is a lambda, PM2-, -6-cystovirus, Acholeplasma, MS2-, Qbeta, Thermoproteusl, OX174-, Spiroplasma-, Mac-l-, T1-, T2-, T3-, T4-, T5-, T6-, T7-, P1-, p2-, MS2-, M20- or S13-phage.

In another preferred embodiment the phage is a filamentous phage.

In an alternative embodiment, during the production of recombinant ZAP® phages the tumour antigen (TA) fusion protein is transported into the periplasmatic space of the bacteria where it gets its native folding as to structure and is then passed into the medium during lysis of the bacteria, since A-bacteriophages do not carry the cpill proteins typical of filamentous phages on the cell surface. In other words, during this process step tumour-specific antigens are expressed as soluble fusion proteins which can be purified, for example, via an affinity column. To facilitate the procedure, a recognition sequence may be attached to the tumour-specific antigen. In a further step the TA fused with cpll can be anchored on the surface of the filamentous phages after in vivo exzision. The genes of the TA are expressed either in fusion with cplll and/or cpVIll. There are 3 5 copies of the cpIll protein on the phage surface.

TA in fusion with this molecules can be present in one or two copies. The coat protein VIII is significantly more frequent on phages (2700-3000 copies) so that TA fused with cpVIII can be present in multiple copies, preferably up to 100 copies on the phage surface.

In a particularly preferred embodiment of the composition according to the invention the filamentous phage is a class I phage, preferably a fd- M13-, fl-, If1-, Ike-, ZJ/Zand Ff-phage or a class II phage, preferably an Xf, a Pfl- and Pf3-phage.

In an additional particularly preferred embodiment the phage coat protein is cpill and/or cpVll or a functionally equivalent fragment thereof. In the present specification and in the present claims the terms "cpIll" and gill" and "cpVIIl" and "gVlll", respectivly, are used synonymously.

In a most preferred embodiment the composition of the present invention, the at least one tumour-specific and/or tumour-associated antigen is expressed as fusion protein with cpVIII or a functionally equivalent fragment thereof and the at least one cytokine as fusion portein with cplll or a functionally equivalent fragment thereof.

In another preferred embodiment the at least one tumour-specific and/or tumourassociated antigen is a single-strand fragment of a lymphoma-specific idiotypic protein or one (or more) antigenic determinant(s) of the idiotypic protein. The singlestrand fragment preferably is an scFv-fragment or a (chimeric) single-chain Fab fragment. The latter can e.g. exhibit human VH and VL chains and a mouse C-part. In a preferred embodiment the rearranged genes of the variable fragments of the immunoglobulin are amplified and connected to a single strand via a linker peptide, see Hawkins et al. 1994 (Blood 83: 3279-88). These are then expressed in fusion with a coat protein gill or gVlll of the phages, preferably VIII. This embodiment allows for the expression of correctly folded, structurally intact idiotypic proteins on the surface of phages.

In another embodiment the present invention relates to a method for the production of the composition according to the invention, wherein the method comprises the following steps: growing of a host cell infected with a phage characterized as above, under conditions which allow for the production of said phage: isolation of said phage from the culture; and optionally combination of the phage isolated in step with a pharmaceutically acceptable carrier and/or diluent.

The phage is isolated from the bacterial supernatant. The concentration generally takes place by means of PEG/NaCI precipitations. By means of multiple precipitations phages can be freed from bacterial contamination very effectively. A further purification step with the aim to eliminate endotoxins from the phage preparation can be carried out by means of several methods, wherein, in principle, there is the possibility of purification via CsCI density. gradient centrifugation, ultracentrifugation, polymyxin B-column chromatography or the TitronX-114 two phase separation and the combination of the various methods. The TitronX-114 two phase separation has proven particularly advantageous which, when carried out several times in sequence, makes a reduction of the endotoxin contamination of initially >300,000 EU/ml to below 10 EU/ml possible, see Aida, Y. et al. 1990 (J.

Immunol. Methods 132: 191-5). Said separation presents a feature of a preferred embodiment of the method of the invention.

In another or preferred embodiment the invention relates to a method for the production of the composition according to the invention which is suitable for the induction of a tumour-specific immune response in Vertebrata, wherein the method comprises the following steps: recovery of nucleic acids from an individual tumour cell, wherein the genes of tumour-specific antigens are amplified from the nucleic acids; cloning, preferably after gel-electrophorectic purification methods and digestion with suitable restriction enzymes, of the genes in a vector system; and expression of the genes in a phage expression system.

Due to the purposeful sequence of the processing steps mentioned above, the production method according to the present invention allows cloning and expression of tumour antigens and antigen receptors on phages within a short period of time. An injection preparation produced according to this method causes, as mentioned before, a polyclonal, antibody-mediated immune response directed against the tumour.

The steps of the method of the present invention for the productiori of an injection preparation are discussed in detail according to a preferred embodiment in the following: The individual tumour cells are obtained from the tumour conglomerate according to standard methods as described e.g. in Sambrook et al., see below.

The nucleic acid, preferably RNA, is isolated and transfered into cDNA and can then be subjected to an RT-PCR-method for fast amplification to multiply the genes of the tumour antigens or fragments thereof.

By means of e.g. a gel-electrophoretic purification method (agarose gel electrophoresis) the DNA fragments are separated according to their size and purified. By means of a connection PCR reaction recognition sequences for restriction endonucleases are introduced into the PCR products to make subsequent cloning possible. After purification of the gene of a tumour-specific antigen e.g. with Geneclean (Bio 101 Inc., La Jolla, CA, USA), the genes are split with different restriction nucleases. The purification and concentration of the DNA fragments is followed by ligation of said DNA fragments in a phagemid vector. This phagemid vector is a vector as e.g. illustrated in Figure 4. In said phagemid vector the gene is preferably cloned N-terminally in the reading frame with the gene of a coat protein.

Preferably, the coat proteins are gill or gVIIl. In a preferred embodiment there is a short Gly-Ser-linker between the proteins. Contrary to traditional phage display vectors, in the present invention and further preferred embodiment(s) preferably vectors are used which do not carry suppressible Amber mutation or its equivalent in front of the coat protein since that could have a negative effect on the presentation density. A recognition signal can be attached N- or C-terminally to the tumour antigen to make its detection or purification possible. Due to the N-terminal fusion of the recombinant protein with a leader sequence like e.g. the OmpA or pELB sequence the transport into the periplasmatic space, where the virus assembly takes place, is made possible. By means of phagemid rescue with helper phages such as M13K07 or VCSM13, hybrid phages are produced carrying the tumour antigens as fusion with a coat protein on their surface. Due to the hybrid order also normal coat proteins, apart from recombinant coat proteins, are present phages, which makes the virus assembly and, thus, the expression of greater proteins on the phage surface possible to start with. The amount of the tumour antigens expressed on the surface can be optimised by induction of the promoter so that the maximal expression is stopped, during which a disruption of the virus assembly has not occurred just yet. Examples of said promoters can be the PLac, the Ptet, PBAo promoter, while other promoters are possible, too.

In an alternative embodiment a A-ZAP®-bacteriophage system is used as vector system, as described in the US patent 5,128,256 (Huse, Sorge, Short). After a packaging process with commercially available packaging extracts GigaPack®, Fa. Statagene) and the amplification of the bacteriophages a transformation in filamentous phages follows by means of co-infection with M13 helper phages. The underlying principle thereby is that the A-bacteriophage vectors carry a plasmid or phagemid vector in them which can be cut out from the phage vector by means of coinfection with helper phages. This step is called in vivo excision and was described for the first time by Short et al. 1988 (Nucleic Acids Res. 16: 7583) and Short and Sorge 1992 (Methods Enzymol. 216: 495-508). An efficient excision of said phagemids is possible with simultaneous co-infection of an E. coli cell with A-ZAP® and filamentous helper phages. After infection of the bacteria the ssDNA of the filamentous phage transducted into the cell is quickly transformed into dsDNA.

Immediately afterwards the expression of phage proteins starts in the host organism, whereby the multiplication of the phages can begin. By means of this method it is possible to express specific tumour antigens for individual tumour cells so that they are present as soluble proteins or as fusion proteins on the phage surface.

In a further preferred embodiment of the method of the present invention the nucleic acids are cytosolic RNAs.

In another preferred embodiment of the method the tumour-specific antigen is fused with a recognition sequence.

Furthermore, the invention relates to the use of a phage characterized as above for the production of a pharmaceutical composition for the induction of a specific immune response, preferably for the specific immune therapy of neoplasia in a Vertebrata.

The invention also relates to methods for the induction of a specific immune response, preferably for the specific immune therapy of neoplasia in a Vertebrata, wherein a phage characterized as above or the composition according to the invention is administered to a Vertebrata.

In a preferred embodiment the neoplasia is a hemoplastosis, preferably a malignant lymphoma or leukaemia, a malignant melanoma, a urogenital carcinoma, preferably a kidney, bladder, prostate or orchic carcinoma, a gyneological carcinoma, preferably a mammary, an ovary, uterus or a cervix carcinoma, a gastrointestinal carcinoma, preferably an esophagus, stomach, colon, rectum, pancreas, gall bladder, biliary duct or hepatocellular carinoma, a malignant endocrinological tumour, preferably a thyroid carcinoma, a malignant lung tumour, preferably a bronchial carcinoma or mesothelioma, a sarkoma or a ZNS tumour.

In another preferred embodiment of the use according to the invention and of the method according to the invention the Vertebrata is a mammal, preferably a human.

Finally, in an additional preferred embodiment of the use according to the invention or of the method according to the invention the pharmaceutical composition is produced as injection preparation, preferably for subcutaneous, intradermal, perinodal, intravenous, peritoneal or intramuscular application or for per os, rectal, intrabronchial or intranasal administration.

The subject matter of these documents is herewith incorporated into the specification by reference.

The figures show: Figure 1: Agarose gel electrophoresis of the PCR products of the idiotypic genes of the Daudi cell line obtainable from the ATCC) with specific primers for the heavy chains (VH and JH primer). Corresponding illustration of the VK and JK primers. For size determination of the fragments the molecular weight marker VI by Boehringer Mannheim was used.

Figure 2: Assembly PCR of Daudi VH and VK fragments. The purified V H and VK fragments are linked to each other during assembly PCR by means of a single chain Fv (scFv) (Gly 4 Ser) 3 linker which is complementary to the 3'-end of the JR gene and the of the VL gene. The markers correspond to the ones of Figure 1.

Figure 3: Western blot analysis of the scFv/cpll fusion proteins with an anti-kappa antibody, which were expressed on filamentous phages, and SDS-PAGE electrophoresis of scFv/cplll after purification via M1 affinity columns. For the M1 affinity column chromatography, the soluble scFv/cplll fusion proteins which were released into the medium from the amplification of recombinant surfZAP® phages were applied in the calcium-containing sample buffer. In the Western blot analysis with anti-kappa antibodies and after M1 affinity chromatography the scFv/cplll fusion protein can be detected as a protein with a size of about 48 kD.

Figure 4: Illustration of the phagemid vectors used in the Examples with Figure 4a representing the surfscript® vector (Stratagene) and Figure 4b representing the cpVIll script.

Figure Cytotoxicity assay: Dentritic cells (DC) pulsed with Bcl2 paghes, Daudi-ld phages and wildtype control paghes were used to induce a primary T-cell response.

Unpulsed DCs were also used as control. The T-cells stimulated by DCs after 48 hrs were added to the tumour cells at various ratios and the cytotoxicity was measured after 3 days. A lysis of Bcl-2 phages could clearly be seen, while Daudi Id phages induce no lysis.

The examples illustrate the invention Example 1: In this Example, the Bcl-2 antigen is cloned and expressed on the surface of the page in fusion with the coat proteins gill and gVIIl. The phagemid vectors used are shown in Figure 4, with a shortened form of the gill protein being used as a gill protein, as has been described in the SurfScript vector (Stratagene). The gVlIl protein consisted of the complete sequence of the protein that can be detected in filamentous phages. It was fused N-terminally in both coat proteins, with the restriction sites Noti and Spel being used for cloning. For the amplification of Bcl-2, the plasmid DNA of the plasmid PB4 from E. coli 79804 (ATCC) was prepared and used in a PCR. Primers for the amplification of Bcl-2 were derived from the Bcl-2 sequence (GenBank M13994) published by Tsujimoto (Tsujimoto and Croce, 1986).

The primers HSBCL2-FOR and HSBCL2-BACK, which were used for the amplification of Bcl-2, correspond to the nucleic acid 1459-1478 or to the reverse complementary sequence 2159-2182 of the aforementioned published article. The amplification of Bcl-2 was carried out in a 50 pI-PCR with 1 x PCR buffer (10 mM Tris-HCL, pH 8.8, 1.5 mM MgCI, 25 Mm KCI) (Stratagene), 0.2 mM dNTPs, 2.5 pmol primer and 2.5 U Taq-polymerase (Boehringer Mannheim) in an OmniGene thermocycler (Hybaid). After 3 minutes of initial denaturation, the Bcl-2 gene was amplified in 30 cycles (1 min 94°C, 1 min 55°C, 1 min 72°C) and in a terminal extension cycle (8 min 72*C). The resulting Bcl-2 PCR product could be shown as a 0.7 kb-fragment in a 1.5%-agarose gel. In a subsequent PCR, restriction sites were inserted so that the product could be cloned into the gill or gVIII phagemid vector in frame with the coat protein. The hybrid phages expressing Bcl-2 were produced as described in the following section on procedures. The expression of the Bcl-2 protein as fusion protein with the coat proteins of the filamentous phage was carried out in a Western blot analysis.

Example 2: In this Example, lymphoma-specific idiotype genes are cloned and expressed on the surface of the phage in fusion with cplll. First of all, cytoplasmic RNA was isolated from the lymphoma cell Daudi according to known methods. Then, the RNA was transcribed into cDNA with (dT),s primers. For one RT-PCR it was sufficient to produce one single-stranded cDNA. The RT-PCR was carried out in an OmniGene thermocycler (Hybaid, c/o MWG-Biotech, Ebersberg) under standard conditions, wherein family-specific human VH and VK primers (cf. Table 1) were used for the amplification. After the RT-PCR, an agarose gel electrophoresis was conducted to analyse the amplification products, i.e. the genes for the heavy chain and the light chain (VL) (cf. Figure After extracting the corresponding VH and VK bands from the agarose gel, the two bands were purified with Geneclean® (Bio 101 Inc., La Jolla, CA, USA) and joined to a single strand (single chain Fv) in an assembly-PCR under conditions as described in the section on procedures. The primers used are listed in Tables 2 to 4. In an assembly-PCR, recognition sequences for the restriction endonucleases are inserted simultaneously into the scFv fragment for subsequent cloning. Then, the product of the PCR was again analysed by means of an agarose gel electrophoresis. Subsequently, the scFv fragments were digested with the respective restriction enzymes and ligated into the g3-script vector. As a comparison, a ligation into the SurfZAP vector (Stratagene) was carried out. The SurfZAP vector was packed in vitro according to the manufacturer's instructions (cf. section on procedures). After in vivo excision (cf. section on procedures), filamentous phage hybrids having Daudi idiotypes on the surface were generated by means of phagemid rescue, as described in the section on procedures. The phage proteins were separated on SDS polyacrylamide gels for the immunochemical detection of the expressed idiotype proteins. In the Western blot analysis, the presence of the expressed idiotype proteins (cf. Figure 2) could be proven. It was also possible to.

purify overexpressed soluble TA fusion-proteins via M1 affinity-columns, with the phages being first diluted 1:1 in a buffer solution (50 mM Tris-HCI, 0.15 M NaCI, 1 mM CaCI 2 5% Triton X-100, pH Then, the pre-equilibrated M1 column (Kodak) was sequentially rinsed several times with 5 ml glycine-HCI solution (0.1 M glycine- HCI, pH 3.0) and finally equilibrated with 5 ml TBS. The affinity column was loaded with the soluble TA in the presence of 1 mM CaCI 2 The column was washed three times with 12 ml TBS/Ca (TBS buffer 1 mM CaCI2) and the proteins bound to the column were eluated six times with 1 ml glycine-HCI buffer. Tris-HCI, pH 8.0 was added for neutralization. After washing with TBS buffer, the eluated proteins were analysed on polyacrylamide ge!.

Example 3: This Example shows the lymphoma-specific idiotype genes of the cell line Daudi expressed on the surface of the phage in fusion with cpVIII. Isolation, amplification and assembly-PCR of the idiotype genes were carried out as described in Example 2. After restriction digestion, the patient-specific scFv obtained in this manner were cloned into the cpVlIl-script phagemid vector and phages were generated via phagemid rescue as described in the section on procedures. The Daudi idiotype proteins expressed on the surface of phages were detected in a Western blot analysis.

Example 4: This Example gives in vitro data by which a cellular immune response triggered by the phage vaccines produced in Examples 1-3 can be detected. Dendritic cells were prepared as described in the section on procedures and pulsed with the phage idiotype on the 5 th day of the re-differentiation. For this reason, first of all dendritic cells (DCs) were split and washed with medium. Then, resuspension was carried out in a DMEM/10% FCS at a concentration of 2 x 106 cells per ml of medium.

Subsequently, 500 pl (5 x 10 s DCs per well were placed on a 24-well plate. For the loading with antigens, they were pulsed with 50-500 pl phage suspension (1 x 1010 1 x 1011 pfu/ml) for several hours (at least 2 hours). The DCs stimulated in this way were used in proliferation assays and cytotoxicity experiments. T-cell proliferation assays were carried out in 96-well plates. In this case, 5 x 104 T-cells and 5 x 103 irradiated dendritic cells per well were used, which corresponds to an effector/stimulation ratio of 10:1. Each experimental set-up was made out in triplicate..

The DCs loaded with antigens were adjusted to a concentration of 1 x 105 cells/ml in Iscove's medium/10% FCS and irradiated with 4000 rad in order to prevent the 3

H-

thymidine incorporation of the proliferating T-cells to be superimposed by potentially proliferating DCs. After irradiation, the DCs were diluted 1:1. Autologous T-cells were adjusted to a concentration of 1 x 106 cells/ml. After 1:1 dilution in Iscove's medium, 100 pl thereof were placed in the respective wells. Wells containing only DCs or Tcells were filled up to 200 pl with 100 pl Iscove's medium. In all wells 10 pl ConA solution (10 pg/mi Iscove's medium) were pipetted to achieve a submaximum stimulation of the cells, excluding the wells which were to exhibit a maximum stabilisation of the T-cells with PHA (50-100 pg/ml). After 48 h, 72 h and 96 h, 25 p 3 H]-tymidine (25 pCi/ml) per well and the incorporation of tymidine was determined for 18 h. Subsequent to these 18 hours, the 96-well plate was deep-frozen at The 96-well plates were harvested in a harvester and the glass fibre membranes were then measured in the Wallac 1460 scintilation counter. For both Bcl-2 phages and Daudi idiotype phages a proliferation of the T-cells was detected. For measuring the cytotoxicity, 1 x 106 T-cells were activated with 5 x 104 antigen-loaded DCs at a ratio of 1:20 (stimulator:target) for 48 hours. As regards T- and stimulator cells, autologous cells taken from the same donor were used. Iscove's medium with U/mi IL-2 and 5% FCS was used as medium. Then, the IL-2-activated T-cells were used for cytotoxic experiments. T-cells and allogenic lymphoma cells (Daudi or the lymphoma cell HOL which is a lymphoma cell that is typical of a CBCC non- Hodgkin lymphoma) were incubated together in different concentrations (10:1, 1:1, Subsequently, the proportion of lysed lymphoma cells were measured applying standard methods. The result can be seen in Fig. 5 and shows that a specific lysis may be induced by Bcl-2 phages, whereas, due to the lacking expression of the histocompatibility antigens on Daudi cells, there is no lysis in the control experiment with Daudi idiotype phages.

Methods: The following methods used by the applicants are described in Sambrook, Fritsch, E.F. and Maniatis, T. 1989, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY: therefore, rapid DNA isolation, restriction digestion, ligation, agarose gel electrophoresis, immunoblotting, bacterial cultures with different amounts media and polyacrylamide gel electrophoresis are not described herein in more detail.

Restriction enzymes and ligases by Boehringer Mannheim and New England Biolabs were used according to the:manufacturer's indications.

The E. coli strain 79804 which carries the plasmid PB4 with the Bcl-2 gene was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Both the universally available cell line Daudi and the lymphoma cell HOL were taken from our culture collection. The experiments can also be carried out or repeated with other cancer cells/cancer cell lines which have corresponding properties and which are available to the skilled person or which he/she can isolate without further ado.

RNA preparation modified according to Favaloro: Lymphoma cells of the cell line Daudi were used. 1-2 cryotubes containing approximately 2 x 107 cells were thawed and diluted with 10ml McCoy medium. The cells were centrifuged at 300 x g and washed three times with 5 ml cold PBS solution (120 mM NaCI, 2.7 mM KCI, 10 mM K-phosphate buffer, pH 7.4, The cell pellet was put into 375 pl RNA extraction buffer (50mM Tris-HCI, pH 8.0, 100 mM NaCI, mM MgCI 2 0.5% Nonidet P-40, 1 mM DTT, 1000 U/ml RNAsin, 4°C) and pipetted up and down until a complete lysis took place. Subsequently, it was incubated in an ice bath, centrifuged and 4 pl 20% SDS was added to the supernatant. Finally, the RNA was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1). By adding 0.1 vol. 3 M Na acetate, pH 5.2 and 2.5 vol. ice-cold ethanol, the RNA was precipitated over night at -20*C or for 30 minutes at -80 0

C.

After centrifugation and one washing step, the RNA pellet was put into 50 pl DEPC-

H

2 0.

cDNA synthesis modified according to Gubler and Hoffmann: For a cDNA synthesis reaction 1-2 pg cytoplasmic RNA were used which was filled up to a volume of 8 p with ddH 2 0 and incubated for 10 minutes at 65 "C in order to remove disturbing secondary structures. Then it was put on ice. At the same time, the cDNA reaction mixture was prepared containing 2 pI reaction buffer (100 mM Tris-HCI, 500 mM KCI, pH 4 pl 25 mM MgCI 2 2 pI 10 mM dNTP mixture, 2 pi oligo-(dT)ls primers (0.8 pg/pl), 50U RNase inhibitor (Boehringer Mannheim) and U AMV reverse transcriptase (AMV-RT, Boehringer Mannheim) per reaction mixture. The denatured RNA was incubated with the cDNA reaction mixture for minutes at 25°C for the oligo-dT primers to hybridize to the RNA. Then, the first strand of cDNA was written during an incubation of 1 hour at 37°C. After the cDNA synthesis, the AMV-RT was deactivated for 5 minutes at 94CC. The single-stranded cDNA was either used immediately in the PCR or stored at -20°C until further use.

Standard conditions for the PCR: For a standard PCR 100 ng matrix DNA were used. Each 100 pl reaction mixture contained 1 x Taq buffer (10 mM Tris-HCI, 50 mM KCI, pH 0.2-0.02 mM dNTP mixture, 1.5 mM (1-2.5 mM) MgCI 2 20 pmol of primers and 2.5 U Taq-polymerase. In the standard reaction 25-30 cycles of 1 min at 94°C (denaturation), 1 min at annealing temperature (melting temperature of the primer minus 5°C) and 1 min at 72°C (polymerization) were carried out. Instead of Taq polymerase also a "proofreading" polymerase such as Pwo-polymerase can be used for amplification of DNA fragments. Pwo-polymerase makes distinctly fewer mistakes.

RT-PCR reaction: 5-20 p cDNA were used in the RT-PCR reaction. With a total volume of 100 pl, the PCR reaction mixture contained 8 pi PCR buffer (identical with cDNA reaction buffer), 2 pl 25 mM MgCl 2 1 p1 gelatine 0.05%, 0.5 pl Taq DNA polymerase (5 U/pl) and pl upstream and downstream primers (20 pmol/pl) per reaction. (The sequences of the primers used are listed in the appendix). The RT-PCR mixture was layered with 2 drops of mineral oil (Sigma Chemical, St. Louis, MO, USA) in order to avoid condensation. The PCR was carried out in an OmniGene thermocycler (Hybaid, c/o MWG-Biotech, Ebersberg) under the following amplification conditions: Initial cycle Denaturation 94 0 C 4 min Amplification cycles, 30 cycles Denaturation 94°C 1 min Annealing 50-60°C 1 min Extension 72°C 1 min Extension cycle Extension 72°C 8 min After the RT-PCR reaction, an agarose gel electrophoresis was conducted to analyse the amplification products. The visibility of weak bands was improved by staining with SYBRTM-Green (FMC Bioproducts). In part, a proof-reading polymerase like the Pwopolymerase was used instead of Taq-polymerase. In this case, MgSO 4 instead of MgCI 2 had to added to the PCR.

Standard conditions for the assembly PCR of idiotvype genes: At least 100 ng of the idiotype PCR fragments and/or the heavy and light chain were used for the assembly PCR since the starting amount of primary DNA fragments is crucial for the results of the assembly PCR. For removing the primers still present, idiotype PCR products were purified with Geneclean (Bio 101 Inc., La Jolla, CA, USA). 100 to 500 ng of the two DNA fragments were used in the reaction. An about equimolar amount of linker primers was used. With a total volume of 50 pl, the assembly reaction mixture contained 5 pl 10 x PCR buffer (100 mM Tris-HCI, 500 mM KCI, pH 3 pl 25 mM MgCI 2 (MgSO4), 100-500 ng each amplified VH and Vm, 2.5 U Taq (Pwo-) DNA polymerase and 2 pl each linker primers (25 pmol/pl) per reaction. The sequences of the linker primers used are listed in Table II. The assembly PCR was layered with 2 drops mineral oil. The amplification conditions for the assembly PCR were as follows: an initial cycle of 94"C 4 min, ten repeating cycles of 94°C 1 min, 60°C 2 min, 72°C 1 min. Immediately after that, a pullthrough PCR with mega primers was carried out to amplify the variable singlestranded fragments (scFv). Degenerated primers were used which flank the scFv on the outside and which additionally contain sequences for restriction endonucleases, a pelB-leader sequence and a flag protein. 50 pl master mixture were prepared for the PCR with said mega primers. This master mixture contained 5 pl 10 x PCR buffer (Stratagene, La Jolla, CA, USA), 5 pl formamide 50%, 1 pi 10 mM dNTPs, 20 pmol each outside primers, 2.5 U Taq (Pwo) DNA polymerase. In this mixture, -the mixture of the assembly PCR was pipetted and layered with mineral oil. This pull-through PCR was carried out under the following amplification conditions: Initial cycle Denaturation 94°C 1 min Amplification cycles, 5 cycles Denaturation 94°C 1 min Annealing 50°C 2 min Extension 72°C 1 min Amplification cycles, 30 cycles Denaturation 94°C 1 min Annealing 55°C 2 min Extension 72°C 1 min Extension cycle Extension 72°C 8 min After the PCR reaction, an agarose gel electrophoresis was conducted to analyse the PCR products fromed.

Packaging of X-bacteriophaqes: For packaging X-bacteriophage DNA packaging extracts by Stratagene, Heidelberg were used. 1 pg ligated phage DNA was incubated briefly on ice in a freeze thaw extract. Then, 15 pl of the thawed Sonic extract were added. The packaging reaction as such was then carried out during a two-hour incubation at 22'C. The reaction was stopped by adding 500 pl SM buffer (100 mM NaCI, 0.2% MgSO 4 50 mM Tris-HCI, pH 7,5, 2% g/v gelatine) and the proteins were precipitated by adding chloroform.

After a brief centrifugation, the supernatant was transferred into a clean Eppendorf vessel and stored at 4 0 C until amplification. After packaging, the titre of the produced infected phage particles was determined.

Amplification of the SurfZAP®-X phages: ml TB medium (LB 0.2% g/v maltose 10 mM MgSO 4 was over-injected with a bacteria colony of a fresh overnight culture and incubated in a shaking incubator until reaching the late midlog stage. After centrifugation the bacteria were resuspended in mM MgSO 4 and an ODoo of 0,5 was adjusted. 600 pl of this bacteria suspension was infected with 5 x 104 packaged phages. For the absorption of the phages, incubation for 10 min at 37°C was carried out. Subsequently, the bacteria were mixed with warm Top agar and plated onto a 150 mm-agar plate. The plated agar plates were cultivated for 6-8 hours at 37'C and then coated with an SM buffer. The agar plates were incubated over night at 4°C while rotating. During this time, the amplified phages diffused in the buffer. It was possible to catch them-by sucking them off. The amplified phages were centrifuged briefly and stored at 4°C adding 0.3% chloroform.

In vivo excision and amplification of SurfScriptO phages in SOLR® cells: For the in vivo excision XL-1 Blue and SOLR cells were cultivated in TB medium.

After the midlog stage had been reached, the bacteria were centrifuged and resuspended in 10 mM MgSO 4 For XL-1 Blue cells an ODeoo of 5.0 and for SOLR cells an OD6oo of 1.0 was adjusted, and the cells were stored at 4 0 C until further use.

The XL-1 Blue cells were infected with SurfZAP® phages. Then, the bacteria were super-infected with 1:1 ExAssistO helper phages. For the absorption of the phages, bacteria were incubated in a shaking incubator at 37°C while shaking gently.

Subsequently, 20 ml LB medium was added to the bacteria und the culture was incubated in a shaking incubator at 37*C at 150 rpm for 2 to 3 hours. After culturing, the bacteria were incubated for 20 min at 70°C, by which the bacteria and X-phages were killed. After centrifugation, the supernatant containing the SurfScript 0 phages was transferred to a fresh tube and stored for 1-2 months at 4CC. Amplification was carried out with SOLR® strains. They were infected at a ratio of 1:1 and incubated in 100 ml LB medium adding 100 pg/ml Carbenicillin and 50 pg/ml Kanamycin until the midlog stage. The bacteria were pelleted by centrifugation and resuspended in mM MgSO 4 Production of competent bacteria (according to a method by Hannahan): The E. coli strain XL-1 Blue was pre-cultivated over night on an LB-agar plate to obtain individual colonies. A colony was multiplied in TYM medium Bacto Trypton, 0.5% YEAST extract, 0.1 M NaCi and 10 mM MgSO 4 until an ODo of This suspension was put into 100 ml TYM and multiplied again until an ODo of Then, the bacteria were cooled to 0°C and centrifuged (15 min at 4000 rpm, 4°C).

The pellet was carefully resuspended in 20 ml TFB-1 solution (30 mM KOH, mM MnCI 2 100 mM CaCI 2 15% glycerine) and incubated for 15 min at 0°C. After centrifugation (3800 rpm/5 min/5°C), the pellet was resuspended in 4 ml TFB-II solution (10 mM MOPS, pH 7, 0.75 mM CaCI 2 10 mM KCI, 15% glycerin) and aliquotted in pre-cooled Eppendorf vessels at 100-200 pl. Then, it was frozen in liquid nitrogen and stored at Transformation of bacteria: 100 pl competent cells were thawed and 2 pl 0.5 M a-ME was added. Part (1-5 pl) of a 20 pl-ligation mixture was carefully added and carefully mixed with the cells.

Subsequently, the mixture was incubated for 30 minutes in an ice bath. Then, the suspension was incubated for 45 sec at 42"C in a water bath (thermo chamber) and again placed in an ice bath for 5 min. 0.9 ml SOB medium was added to the suspension, then it was shaken for 60 min at 37°C. An aliquot of the mixture (200 pl) was plated onto a pre-warmed LB-agar plate plus Ampicillin and incubated over night at 37*C. Clones grown in this way were multiplied and analysed by a standard rapid disruption method and subsequent restriction digestion.

Phaqemid Rescue: Filamentous hybrid phages were generated carrying the tumour antigens on the surface in fusion with a coat protein. TG1-clones were incubated on Minimal agar plates 100 pg/ml Ampicillin) over night at 37°C. In the morning, an inoculating loop containing bacteria of the plate was diluted in 50 ml 2 x YT-AG medium (2x YT medium 100 pg/ml Amp and 2% glucose) and cultivated until the midlog stage or until an OD550 nm of 0.5. When the cells were in the midlog stage (OD550 nm of M13K07 or VCSM13 helper phages were added with a MOI of 1:10 to the bacteria.

The bacteria were incubated for 30 min at 37°C while shaking gently. The bacteria were centrifuged and resuspended in 500 ml 2 x YT-AK (2 x YT medium 100 pg/mi Amp and 50 pg/ml Kanamycin). IPTG was added at the desired concentration (normally 0.5-2 mM/ml) and the bacteria were cultivated for 18 h in a shaker at 180 rpm at 300C. Then, the bacteria culture was centrifuged at 4500 x upm for minutes at 4*C. The supernatant was transferred to a 1000 mi-vessel and the phages were precipitated over night in an ice bath adding 1/5 vol. (125 ml) PEG/NaCI. The phages were then transferred to 250 mI-Corning centrifuge tubes and centrifuged at 4000 rpm for 15 min at 4*C. The phage pellet was resuspended in 30 ml 1 x PBS and transferred to Oak-Ridge centrifuge tubes (Nalgene 3115-0050) and centrifuged for min at 15,000 rpm at 4°C in an SS34 rotor. Thereby, the remaining cell components of the bacteria were pelleted. The phages were then precipitated once more adding 1/5 vol. PEG/NaCI and resuspended in 5 ml PBS. For pasteurization, the phages were filtered through a 0.45 pm-sterile filter and stored at 4"C until use or aliquots of it were deep-frozen at -200C.

Endotoxin purification of phages by means of Triton X-114: The phage suspension was sterile filtered and precipitated with 1/5 vol.

PEG8000/1.5M NaCI. The phages were resuspended in 1 ml PBS. 1% Triton X-114 was added. 5 min-incubation on ice until the suspension appears to be homogenous.

The sample was incubated for 5 min at 37*C and briefly centrifuged at max. speed in a standard Eppendorf centrifuge. As a result, 2 phases were formed, the top aqueous phase containing the phages. This phase is carefully sucked off without contaminating the bottom phase. This Triton X-114 two-phase separation is repeated three times with the aqueous phase. Finally, the phages are again precipitated with PEG/NaCI and the phages were resuspended in PBS. The endotoxin content was determined using the LAL method, with the endotoxin content normally being in a range of 0.1 to 10 EU/ml. The Triton X-114 two-phase separation is to be repeated until the desired degree of purity has been reached.

Isolation of leukocytes from the peripheral blood: After a blood sample from the vein was taken, 5 ml each of heparin blood was transferred to a 50 ml-Falcon® tube and layered with 40 ml of cold Ery-lysis buffer (155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, 0.2 mM EDTA). During the lysis, the solution was incubated for 10 min at 4°C. Then, the suspension was centrifuged (1280 rpm, 10 min, 4*C) and washed three times with PBS Dulbecco's (Gibco/BRL, Grand Island, NY, USA). The pellet was resuspended in medium and a concentration of 1 x 107 cells/ml was adjusted. Until further use, the cells were stored in liquid nitrogen adding 20% FCS and 10% DMSO, or they were used immediately.

Isolation of mononucleic cells by means of density gradient centrifugation: Heparin blood was diluted 1:1 in PBS. 20 ml Ficoll separating solution (Seromed, Biochrom AG, Berlin), which had a temperature of 20"C was put in a 50 mi-Falcon® tube and carefully layered with 20 ml PBS blood. After centrifugation (2000 rpm, min, room temperature, without brake), the inter-phase (containing monocytes, lymphocytes and blasts) was carefully sucked off with a Pasteur pipette. The mononuclear cells were washed twice in ice-cold PBS and either processed immediately or deep-frozen in freezing medium (450 pl RPMI, 450 pl McCOY,

DMSO).

Magnetic cell separation: For the magnetic cell separation, the cells were incubated with a sufficient amount of specific antibodies as regards separation (10-20 pIl/ x 107 cells) in 200 pl PBS/BSA at 4-8 0 C for 30 min. For removing the surplus antibodies, the cells were washed three times with PBS/BSA (2000 rpm, 4 min, RT). Then, the cells stained with the primary antibody were put in 100 pl PBS/BSA and incubated with a second ferrochrome-containing secondary antibody (25 pl/1 x 10 7 cells) for 30 min at 4*C.

After washing again, the cells treated in this way were applied to a pre-equilibrated RS column hanging in magnetic field (Milteyni-Biotech) so that the labelled cells were magnetically fixed to the column. The ceils fixed in that way were washed in a sufficient amount of PBS/BSA while the unfixed cells were collected in a different tube for further use. After removing the magnet, the magnetically fixed cells could be eluted from the column by washing with PBS/BSA.

Generation of dendritic cells: For generating dendritic cells, monocytes from a buffy coat or from periphery blood were used. After density gradient centrifugation with Ficoll solution (see above), the mononucleic cells were adjusted to a concentration of 5-10 x 106 cells/ml in Iscove's medium (without serum). 15 ml cells were then pipetted in cell culture flasks and cultivated in an incubator for one hour in order to separate monocytes from lymphocytes. During this time, the monocytes adhere to the bottom of the culture flasks. Subsequently, the lymphocytes that do not adhere to the bottom of the culture flasks were sucked off and used for preparing the T-cells. The adhering monocytes were washed several times with warm medium and incubated again in an incubator for one hour. After washing again, the adhering cells were put in RPMI 1640 medium containing 5% FCS or 1% autologous serum. Normally, the monocytes .were enriched to about 60% in this way. For obtaining a more pure CD14* population, the adhering monocytes were purified via antibodies coupled with a magnet by means of MACS (Miltenyi-Biotech, Bergisch Gladbach) to a degree of more than For the redifferentiation into dendritic cells, the monocytes were cultivated in RPMI 1640 medium adding Pen/Strep and L-glutamine at 37 0 C, 5% CO2. 800 U/ml GM- CSF and 500 U/ml IL-4 (both cytokines by Genzyme) was added to the medium.

Every 2-4 days, the medium and the cytokines were renewed. The purity of the preparation and the steps of the redifferentiation into dendritic cells were controlled by FACS analyses.

Further methods which are not described in more detail herein, bacteria strains, phage strains, and other material are of conventional origin or can be bought.

Table I: Primer for the polymerase chain reaction (PCR) A. Primer for the amplification of the variable fragment of the heavy chain: 1. VH primer: HSVH IA HSVH 2A HSVH 3A HSVH 4A HSVH 5A HSVH 6A CDR RVG CAR CTK GTG CAR TCT GG -3' CAG GTN CAR CTG SWG NAG TCK GG -3' SAG GTN CAG STG GTG SAG TCN G -3' CAG GTG CAG CTG CAG SAG TCG -3' SAR STG CAG CTG KTG CAG TCV G -3' CAG GTA CAG CTG CAG CAG TCA -3' 2. Cp primer:

HSIGM-CH

3. JH primer: HSJH-12FOR HSJH-3FOR HSJH-6FOR AAA GGG TTG GGG CGG ATG CAC TCC -3' TGA GGA GAC GGT GAC CAG GGT GCC -3' TGA AGA GAC GGT GAC CAT .TGT CCC -3' TGA GGA GAC GGT GAC CAG GGT TCC -3' TGA GGA GAC GGT GAC CGT GGT CCC -3' B. Primer for the amplification of the variable fragment of the K-light chain: 1. V, Primer: HSVK IA HSVK 2A HSVK 3A HSVK4A HSVK 5A HSVK 6A HSVK 7A RHC ATC VRG ATG ACC CAG TCT CC -3' GAW RTT GTG WTG ACN CAG WCT CCA -3' GAA ATW GTR WTG ACR CAG TCT CCA -3' GAC ATC GTG ATG ACC CAG TCT CCA -3' GAA ACG ACA CTC ACG CAG TCT CCA -3' GAW RTT GTG MTG ACW CAG TCT CCA -3' GAC ATT GTG CTG ACC CAG TCT CCA -3' 2. J, Primer: HSJK 124FOR HSJK 3FOR HSJK 5FOR ACG TTT GAT CTC CAC CTT GGT CCC -3' ACG TTT GAT ATC CAC TTT GGT CCC -3' ACG TTT AAT CTC CAG TCG TGT CCC -3' C. Primer for the amplification of the variable fragment of the I-light chain: 1. Vx primer: HSVL 1A HSVL 2A HSVL 3A HSVL 4A 2. Jx primer: HSJL 1FOR HSJL 23FOR HSJL 7FOR CAG TCT GTG TTG ACG CAG CCG CCC TC -3' CAG TCT GCC CTG ACT CAG CCT GCC TC -3' TCC TAT GAG CTG ACT CAG CCA CVC TC -3' CAC GTT ATA CTG ACT CAA CCG CCC TC -3' ACC TAG GAC GGT GAC CTT GGT CCC -3' ACC TAG GAC GGT CAG CTT GGT CCC -3' ACC GAG GAC GGT CAG CTG GGT GCC -3' Table II: Primers for assembling light and heavy chains to form a variable singlestrand fragment (scFv) (Gly 4 Ser) 3 linker sequence: GGT GGC GGT GGC TCG GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT reverse (Gly 4 Ser) 3 linker: AGA TCC GCC GCC ACC CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC scJH linker: scHSJH-12FOR scHSJH-3FOR scHSJH-6FOR scVL linker: scHSVK-1 A scHSVK-2A scHSVK-3A scHSVK-4A scHSVK-6A scHSVK-7A CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC TGA GGA GAC GGT GAC CAG GGT GCC -3' CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC TGA AGA GAC GGT GAC CAT TGT CCC -3' CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC TGA GGA GAC GGT GAC CAG GGT TCC -3' CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC TGA GGA GAC GGT GAC CGT GGT CCC -3' GGC GGT GGT GG G GT G GGT GGC GGC GGA TCT RHC ATC VRG ATG ACC CAG TCT CC -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT GAW RTT GTG WTG ACN CAG WCT CCA -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT GAA ATW GTR WTG ACR CAG TCT CCA -3' GGC GGT T G GGG TCG GGT GGC GGC GGA TCT GAC ATC GTG ATG ACCCAG TCT CCA -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT GAA ACG ACA CTC ACG CAG TCT CCA -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT GAW RTT GTG MTG ACW CAG TCT CCA -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT GAC ATT GTG CTG ACC CAG TCT CCA -3' scHSVL-1A scHSVL-2A scHSVL-3A scHSVL-4A GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT CAG TCT GTG TTG ACG CAG CCG CCC TC -3' GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT CAG TCT GCC CTG ACT CAG CCT GCC TC -3' GGC GGT GOT GGG TCG GGT GGC GGC GGA TCT TCC TAT GAG CTG ACT CAG CCA CVC TC -3' GGC GGT GOT GGG TCG GGT GGC GGC GGA TCT CAC GTT ATA CTG ACT CAA CCG CCC TC -3' Table IIl: Degene sequen SZ-pel B-F LAG-H S- CTA TTG COT AC GCA ATG GOC G SZ-pelB-FLAG-HSr- CTA TTG COT AC GCA ATG GOC G SZ-pelB-FLAG-H.'S CTA TTG COT AC GCA ATG GCO G SZ-pelB-FLAG-HSr- CTA TTG COT AC GCA ATG GOC G SZ-pel B-F LAG-H S.

CTA TTG COT AC GCA ATG GOC G, SZ-pelB-F LAG-H S CTA TTG OCT AC GOA ATG GOC G, rated primers with peIB-leader and FLAG& Taq reoognition VH-1 primer: G GOG GCC GCA GGT CTC OTO OTO TTA GOA GCA C O TAO AAA GAO GAT ODR RVG OAR OTK GTG CAR I VH-2 primer: G GOG GOC GCA GGT OTO OTO OTO TTA GCA GCA C TAO AAA GAO GAT CAG GTN OAR OTG SWG NAG' VH-3 primer: G GOG GC GCA GGT OTO OTO OTO TTA GOA GCA C TAO AAA GAO GAT SAG GTN CAG STG GTG SAG I VH-4 primer: G GOG GOC GCA GGT OTO OTO OTO TTA GOA GCA C TAO AAA GAO GAT CAG GTG CAG OTG CAG SAG I VH-5 primer: G GOG GOC GCA GGT CTC OTO OTO TTA GOA GCA C kO TAO MAA GAO GAT SAR STG CAG OTG KTG CAG T VH-6 primer: G GOG GOC GCA GGT OTO OTO OTO TTA GOA GOA C kC TAO MAA GAO GAT CAG GTA CAG CTG CAG GAG I AA OCA CT GG AA OCA FOK GG AA COA ON G AA COA

"CG,

AA OCA CV G

AAOCCA

'CA

Table IV: Primers with Spel cleavage site SZ-FX-HSJK/L primer: AGCATCACTAGT (CTA)-HSJK/L primer HSJK 124FOR AGCATCACTAGT (CTA) ACG TTT GGT CCC -3' HSJK 3FOR AGCATCACTAGT (CTA) ACG TTT GGT CCC -3' HSJK 5FOR AGCATCACTAGT (CTA) ACG TTT TGT CCC -3' HSJL 1FOR AGCATCACTAGT (CTA) ACC TAG GGT CCC -3' HSJL 23FOR AGCATCACTAGT (CTA) ACC TAG GGT CCC -3' HSJL 7FOR AGCATCACTAGT (CTA) ACC GAG GGT GCC -3' GAT CTC CAC GAT ATC CAC AAT CTC CAG

CTT

TTT

TCG

GAC GGT GAC GGT GAC GGT GAC CTT CAG CTT CAG CTG The entire disclosure in the complete specification of our Australian Patent Application No. 41442/99 is by this cross-reference incorporated into the present specification.

Claims (18)

1. A vaccine comprising a bacteriophage or a functionally equivalent fragment thereof which expresses on its surface at least one tumour-specific and/or tumour-associated antigen having a minimum length of 20 amino acids as a fusion protein with a phage coat protein cpVIII or a fragment or a functionally equivalent derivative thereof, and which optionally contains a pharmaceutically acceptable carrier and/or a dendritic cell and/or a vehicle and/or a pharmaceutically acceptable diluent.

2. The vaccine according to claim 1, wherein the phage or the functionally equivalent fragment thereof co-expresses on its surface at least one cytokine as a fusion protein with the phage coat protein or the functionally equivalent derivative thereof.

3. The vaccine according to claim 1 or 2, wherein the bacteriophage is a filamentous phage.

4. The vaccine according to claim 3, wherein the filamentous phage is a class-I phage, preferably an fd, M13, fl, If1, Ike, ZJ/Z or Ff phage, or a class-ll phage, preferably an Xf, Pfl or Pf3 phage. The vaccine according to any one of claims 2 to 4, wherein the at least one tumour-specific and/or tumour-associated antigen is expressed as a fusion protein with cpVIII or a functionally equivalent fragment thereof and the at least one cytokine is expressed as a fusion protein with cplll or a functionally equivalent fragment thereof.

6. The vaccine according to any one of claims 1 to 5, wherein the at least one tumour-specific and/or tumour-associated antigen is a single-stranded fragment of a lymphoma-specific idiotype protein or an antigenic determinant thereof.

7. A method for the production of a vaccine according to any one of claims 1 to 6 comprising the following steps: 36 H:UuanitaWKeeppa ten41442-99.DIV.doc 13/11/03 cultivating a host cell which has been infected with a phage as defined in any one of claims 1 to 6 under conditions allowing the production of said phage; isolating said phage from the culture; and optionally combining the phage isolated in step with a pharmaceutically acceptable carrier and/or diluent.

8. The method according to claim 7 for producing a vaccine according to any one of claims 1 to 6 which is suitable for inducing a tumour-specific immune response in vertebrates, the method comprising the following additional steps: recovering nucleic acids from a tumour cell, wherein the genes of tumour-specific antigens are amplified from the nucleic acids; cloning the genes into a vector system, preferably after gel electrophoretic purification methods and digestion with suitable restriction enzymes; and expressing the genes as phage fusion proteins.

9. The method according to claim 8, wherein the nucleic acids are RNAs. The method according to claim 8, wherein the nucleic acids are DNAs.

11. The method according to claim 9, wherein the RNAs are cytosolic RNAs.

12. The method according to any one of claims 8 to 10, wherein the tumour- specific antigen is fused with an recognition sequence which allows the detection and/or purification of the antigen.

13. Use of a phage as defined in any one claims 1 to 6 for producing a pharmaceutical composition for inducing a cytotoxic T-cell immune response for a specific immunotherapy of a neoplasia in a vertebrate. 37 H:UuanitalKeep\patentI 1442-99.DIV.doc 13/11 03

14. Use according to claim 13, wherein the neoplasia is hemoblastosis, preferably a malign lymphoma or a leukemia, a malign melanoma, a urogential carcinoma, preferably a kidney, bladder, prostatic or testicular carcinoma, a gynecologic carcinoma, preferably a mammary, ovarian, uterine or cervix carcinoma, a gastrointestinal carcinoma, preferably an esophageal, gastric, colon, rectum, pancreas, gallbladder, bile duct or hepatocellular carcinoma, a malign endocrinologic tumour, preferably a thyroid carcinoma, a malign lung tumour, preferably a bronchial carcinoma or mesothelioma, a sarcoma or CNS tumour. Use according to claim 13 or 14, wherein the vertebrate is a mammal, preferably a human.

16. Use according to any one of claims 13 to 15, wherein the pharmaceutical composition is produced in a form suitable for use as an injection preparation for parenteral, preferably subcutaneous, intradermal, intramuscular, perinodal, intravenous or intraperitoneal administration, or for per os, preferably oral, rectal, intrabronchial or intranasal administration.

17. A method of inducing a cytotoxic T-cell immune response for a specific immunotherapy of a neoplasia in a vertebrate comprising administering to a patient in need thereof an effective amount of a vaccine according to any one of claims 1 to 6.

18. A method according to claim 17, wherein the neoplasia is hemoblastosis, preferably a malign lymphoma or a leukemia, a malign melanoma, a urogential carcinoma, preferably a kidney, bladder, prostatic or testicular carcinoma, a gynecologic carcinoma, preferably a mammary, ovarian, uterine or cervix carcinoma, a gastrointestinal carcinoma, preferably an esophageal, gastric, colon, rectum, pancreas, gallbladder, bile duct or hepatocellular carcinoma, a malign endocrinologic tumour, preferably a thyroid carcinoma, a malign lung tumour, preferably a bronchial carcinoma or mesothelioma, a sarcoma or CNS tumour.

19. A method according to claim 17 or claim 18, wherein the vertebrate is a mammal, preferably a human. 38 H:UuanllalKeeppatenMI1442-99.DIV.doc 13/11/03 A method according to any one of claims 17 to 19, wherein the vaccine is provided as a pharmaceutical composition in a form suitable for use as an injection preparation for parenteral, preferably subcutaneous, intradermal, intramuscular, pennodal, intravenous or intraperitoreal administration, or for per os, preferably oral, rectal, intrabrochinal or intranasal administration.

21. A vaccine according to any one of claims 1 to 6, substantially as herein described with reference to the description and figures.

22. A method according to any one of claims 7 to 12 or 17 to substantially as herein described with reference to the description and figures. Dated this 13th day of November 2003. IMMUNOGENEC BIOTECHNOLOGIE GMBH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 39 H:UuanitalKeep'patenM1442-99.DIV.doc 13/11/03