Multi-component tumour vaccine

The present invention relates to the field of tumour vaccine and cancer therapies.

Cancer is the second leading cause of death in the developed countries. In particular, metastatic disease stages of cancer are not or not sufficiently amenable to standard cancer therapy modalities such as surgery, radiotherapy or chemotherapy. These metastases, not the primary tumour, are responsible for the majority of deaths of cancer patients. Immune cancer therapy has become a new modality of cancer treatment aiming at activation of the patient's own immune system to combat the tumour. In particular, elimination of circulating tumour cells and eradication of micro- and macro-metastases which may have remained after surgery or radiotherapy are important targets of cancer immune therapy. Within "passive immune therapy", immune effector molecules (e.g. antibodies, or cytokines) or immune cells have been used to treat cancer patients. Alternatively active immunization ("anti-cancer vaccination") aims to help the patients immune system to recognize tumour cells as foreign and to activate the immune system to eliminate the tumour cells.

It has been shown that tumour-associated antigens (TAAs) can be recognized by T-cells or antibodies resulting in tumour cell destruction. The identification and selection of appropriate TAAs and their presentation to the immune system in a highly immunogenic manner is of utmost importance for the efficacy of active cancer immune therapy. A variety of tumour-associated antigens (TAAs) has been identified as potential targets for cancer immune therapy, including CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, gastrin, CA125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs etc..

Furthermore, carbohydrate and mucin antigens (which are exposed on the cell surface) resulting from aberrant glycosylation of tumour cells distinguishing cancer cells from normal cells have been demonstrated to represent attractive targets for immune therapy of cancer. Although modifications differ from one tissue to another, it has been observed that a modified glycosylation is a typical feature of cancer cells. Examples of tumour-associated carbohydrate structures are the blood group related Lewis antigens which are broadly expressed in many epithelial types of cancer. They include Lewis X, Lewis B and Lewis Y structures as well as sialylated Lewis X structures. Other relevant carbohydrate antigens are GloboH structures, KHl, Tn antigen, TF antigen, alpha- 1,3 -galactosyl epitope, the SialylTn antigen as well as gangliosides, such as GD2, GD3, GMl and GM2.

The SialylTn carbohydrate antigen - as an example of a prominently expressed carbohydrate TAA - is expressed in more than 80% of cancers of breast, colorectal, prostate and ovarian origin and its expression level has been shown to correlate with a more aggressive tumour phenotype resulting in poor prognosis.

However, there are a variety of obstacles for the use TAAs for anti-cancer vaccines. A major limitation to the use of TAAs is their low immunogenicity, or even lack of immunogenicity. In particular, carbohydrate TAAs - as thymus-independent antigens - are not able to induce T-cell help and completely fail to induce memory. The conjugation of the "immunologically inert" carbohydrate structures with thymus-dependent antigens such as proteins, will enhance their immunogenity. Therefore, vaccines based on tumour-associated carbohydrate structures are coupled to so-called "carrier molecules" in order to enhance their immunogenity. Proteins like bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) often serve as carrier molecules. The carrier protein will stimulate the carrier-specific T-helper cells which will then play a role in the induction of the anti-carbohydrate antibody synthesis.

Another option to induce an immune response to carbohydrates is the immunization with so- called "mimotopes" which are by their molecular structure no carbohydrates (e.g., peptides; Nat. Biotechnol. (1999), 17:660; Nat. Biotechnol. (1997), 15:512). Alternatively, ways of inducing an immune response against non-imunogenic TAAs is the use of anti-idiotypic antibodies as immunogens which imitate the structure of a TAA thus triggering an immune response that will also react with the relevant TAA or the fusion of TAAs with specific foreign protein sequences (U.S. Pat. No. 5,869,057, U.S. Pat. No. 5,843,648 and U.S. Pat. No. 6,069,242).

However, another major limitation to anti-cancer vaccination is the heterogeneous expression of TAAs on cancer cells with only a part of the cells in the tumour or tumour type expressing the particular antigen. Tumours use this mechanism for escape from the immune system. Therefore, to minimize the risk of escape variants it is necessary to target more than one antigen for maximal eradication of tumour cells.

Another severe limitation to the currently applied cancer immune therapy approaches is the fact that cancer is often associated with an increasingly impaired immune response against the tumour. Escape mechanisms of the tumour such as loss of antigen expression, reduced MHC expression, absence of co-stimulatory molecules, suppression of anti-tumour immune responses by CD4+CD25+ T regulatory (Treg) cells, and alterations in the cytokine profile - often skewed

to wards a Th2 response - affect the ability of the immune system to mount an effective response against the growing tumour. Therefore, the use of appropriate target antigen(s) together with a Thl/Th2 balanced cytokine profile may be essential to increase therapeutic efficacy. First hints of efficacy of this approach were already provided by the application of Coley's toxin - a bacterial extract administered directly into the tumour - which resulted in marked alterations in cytokine levels and dramatic anti-tumour responses, but also significant toxicities in patients. Application of recombinant cytokines also has been shown to be associated with severe side effects and therapeutic effects have been shown in only few cases so far. This limited efficacy may be due to the limitation in simulating correctly the biological paracrine function of cytokines in the context of antigen uptake and presentation. Furthermore, the synchronized, often synergistic, action of various cytokines rather than single cytokines may be required for optimal induction of an immune response. Proof of principle for the "paracrine action" of cytokines has been demonstrated using cytokine gene-modified tumour cells derived from autologous tumour material of the patient. However, the approach of using autologous tumour material is very cumbersome and is not amenable to large scale drug development and will not be adaptable for treatment of large patient populations. For development of anti-cancer drugs applicable to large patient populations, molecularly defined synthetic vaccines which combine an immunogenic presentation of relevant TAAs with the ability to provide a balanced Thl/Th2 cytokine profile, and which are amenable to large scale pharmaceutical manufacturing process are necessary.

The WO 1996/34005 Al describes synthesis of immunogenic oligonucleotides bound to an adjuvant in order to induce an immune response. Suitable adjuvants are e.g. protein carriers, bacteria, liposomes and bacille Calmette-Guerin.

The WO 2005/004809 A2 describes multivalent antibodies which can bind multiple epitopes including epitopes of tumour associated antigens. The arms of the antibody may bind the target epitope and additionally a carrier e.g. peptides, peptide derivatives, oligopeptides and oligonucleotides.

The EP 674 907 A2 relates to conjugates of antibodies, e.g. anti-idiotypic antibodies, and a carrier molecule. The anti-idiotypic antibody may mimic tumour associated antigens. The carrier can be immunogenic itself, e.g. in the case of bacterial toxines. Each carrier molecule may bind only one antibody.

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The WO 2003/097663 describes an immunogenic monoclonal antibody which comprises at least two different epitopes of a tumour-associated antigen. The antibody may also be specific for such a tumour-associated antigen e.g. in the case anti-idiotypic antibodies to increase the immune response.

The object of the present invention is to avoid the drawbacks of the currently available tumour vaccines as described in the prior art, and to provide a method for generating an improved immune response against tumour cells.

According to the invention, this object is achieved by providing a pharmaceutical multicomponent anti-tumour preparation comprising

(i) epitopes of at least two tumour-associated antigens,

(ii) at least one immunogenic carrier,

(iii) at least one adjuvant, preferably coformulated, in a slow release formulation. Such a preparation can be used to generate a immune response against tumour cells in combination with the activation of antigen unspe- cific effector cells with anti-tumour activity. By using tumour-associated antigen epitopes, such as peptides, proteins, oligosaccharides, glycoproteins or glycosylated peptides (i.e. antigens resulting from aberrant glycosylation) coupled to an immunogenic carrier molecule resulting in a "tumour-antigen-carrier conjugate" comprising epitopes of at least two different tumour-associated antigens in the slow-release formulation comprising both, the molecular conjugate and a adjuvant in a highly immunogenic formulation resulting in a "multi-component tumour vaccine" is obtained which has the potential to even activate antigen non-specific effector cells.

The inventive slow release formulation of the epitopes and the immunogenic carrier together with the adjuvant leads to an increased activation of the immune system localized at the area of application of the inventive preparation. In particular, in the case of cancer by topical administration to a subject can thus lead to increased activation and accumulation of antigen specific and supportive immune cells. Such slow release formulations may include the co- formulation of the antigens/carriers with slow-release mediators such as liposomes, polymeric microspheres, dextran, cationic lipids, nano microparticles. In particular, preferred embodiments the preparation is formulated with aluminium hydroxide, optionally after heat adhesion or by

coformulation with slow-release mediators resulting in co-adsorption of the antigen/carrier/adjuvant components. Aluminium hydroxide is usually used as weak adjuvant. By heat treatment of aluminium hydroxide with an antigen, the adhesion is increased which leads to a substantially slower desorption. In principle, any adjuvant together with an antigen can be turned into a slow release formulation by heat treatment (US 2003/0143221 Al). In case of aluminium hydroxide as slow-release mediating agent, it was shown that for co-heat treated antigen the desorption by incubation for 30 min by 37°C (as comparison) in a bovine serum albumin solution was reduced to 31% after treatment at 6O ⁰ C for 30 min, to 7% after heat treatment at 80°C for 30 min, and even < 1% after heat treatment at 121°C for 30 min in an autoclave as compared to non-heat treated preparations. A simple adsorption of only the antigen/carrier composition onto aluminium hydroixde does not qualify for the inventive characteristics of the slow release formulation according to the present invention which requires the coformulation and thus the coadsorption with the adjuvant. Other slow-release formulations comprise nano or micro particles of e.g. granules which contain an inventive antigen/carrier. The granule matrix can be for example comprise more than one layer carrying an inner layer with the antigens and/or carriers (e.g. US 4 880 830). Further slow release formulations include slow solving materials which incorporate the antigens in the carriers. The formulation of the preparation in form of particles, e.g., in the range of 10 nm to 10 μm, preferably 100 nm to lμm, increases the uptake of the antigens in antigen-presenting cells (for example dendritic cells, microphages) and subsequently, the induction of the immune reaction. These particles can e.g. be formed by co-formulation and adsorption of the components with particulate slow-release mediators, such as particulate aluminium hydroxide.

Preferably, the immunogenic carrier is an immunogenic protein, most preferably an anti- idiotypic antibody or a mimotopic protein, e.g. a mimotopic antibody, which imitates the structure of at least one epitope of a TAA, thus triggering an immune response that will also react with this TAA.

Preferably, epitopes of tumour-associated antigens ("TAAs") selected from the group consisting of peptides or proteins, oligosaccharides, glycoproteins, glycosylated peptides or of an aberrant glycosylation of tumour cells. Particularly preferred epitopes are of the TAAs CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, CA125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs etc and T-cell peptides preferably derived from tumour-associated

antigens, or of carbohydrates, particularly JLewis Y, Tn, TF, SialylTn, GloboH, and gangliosides, such as GD2, GD3, GMl and GM2. The epitopes are coupled to the immunogenic carrier, giving raise to a tumour-antigen-carrier conjugate which comprises epitopes of at least two TAAs.

According to the invention the tumour-antigen-carrier conjugate is formulated in a highly immunogenic way in the slow-release formulation to give raise to a multi-component anti- tumour preparation. Preferably, the preparation comprises or is co-formulated with additional adjuvants in order to induce a strong immune response and to elicit immune stimulatory cytokines, upon administration, such as IFNγ, IL-2, IL- lβ, TNFα, IL-4, GM-CSF, IL- 12, IL-6, IL- 12 leading to an additional activation of antigen unspecific effector cells. Quantitatively, the increase of TNFα, IL- lβ or IL-2 upon administration in a subject, e.g. a mammal such as a human or a combination of at least two of IL-4, GM-CSF or IL- 12 is at least by 10 pg/ml, preferably at least 100 pg/ml, serum or plasma, or of IFNγ by at least 100 pg/ml, preferably at least 1000 pg/ml, serum or plasma.

Preferably the preparation is a vaccine. A vaccine is in form of a composition which can be administered to a subject, e.g. a mammal such as a human, dog or horse, and upon administration stimulates an immune response against a tumour. Subsequently, the immune system will attack the tumour and reduce its activity or substance. A therapy with such a vaccine can be used in a therapy against cancer or as prophylactic measure to reduce the chance of developing the tumour.

Surprisingly it has been shown in the present invention that injection of an immunogenic formulation of a tumour-antigen-carrier conjugate vaccine composed of, e.g. SialylTn carbohydrate tumour-associated antigen epitopes coupled to a highly immunogenic (for humans or Rhesus monkey hosts) murine IgG2a antibody, coformulated with a strong adjuvant, such as QS-21 and coadsorbed onto aluminium hydroxide particles, into Rhesus monkeys, induces (i) a strong immune response against the carrier molecule, (ii) a significant immune response against the SialylTn carbohydrate antigen, (iii) a significant immune response against SialylTn positive OVCAR-3 tumour cells, (iv) antibody-dependent cellular cytotoxicity (ADCC) against SialylTn positive OVCAR-3 tumour cells. Furthermore, a significant immune response also against SialylTn negative KATOIII tumour cells was found, which seems to be related to the immune response against the carrier molecule. Furthermore, it was surprisingly found that application of

this vaccine formulation induces also a cytokine release, especially of IFNγ, IL-2, IL- lβ, IL-4, GM-CSF, TNFα, IL- 12, IL-6 measurable in the serum of immunized animals which was found to correlate with activation of antigen-unspecific effector cells, such as NK cell and other PBMC (peripheral blood mononuclear cells) leading in significant anti-tumour activity (tumour cell lysis) against tumour cells that do not express the SialylTn immunization antigen.

The term "immunogenic" is meant to encompass any structure that leads to an immune response in a specific host system. A xenogenic protein (i.e. a protein derived from another species) e.g. a murine antibody, or fragments of this antibody, will, for instance, exhibit a very strong immunogenic action in another mammal e.g. in monkey or human organisms, which action will be further enhanced by a combination with adjuvants.

The term "epitope" defines that region within a molecule (antigen), which can be recognized by a specific antibody or by a specific T-cell, or which induces the formation of specific antibodies or specific T-cells against this epitope. Epitopes may be conformational epitopes or linear epitopes. Epitopes, above all, imitate or comprise domains of a natural, homologous or derivatized tumour-associated antigen, TAA. Often they are comparable to TAAs at least by their primary structures and, possibly, secondary structures. Yet, epitopes may also completely differ from TAAs in this respect and imitate components of TAAs, such as carbohydrate antigens, merely by the similarity of spatial (tertiary) structures. Thus, the tertiary structure alone, of a molecule is able to form a mimicry ("immunological imitation") which induces an immune response to a specific TAA.

The epitopes of the tumour-antigen-carrier conjugate according to the invention preferably include epitopes of antigens selected from the group consisting of peptides or proteins, particularly CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, CA 125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs etc., and T-cell peptides, preferably derived from tumour-associated antigens, or of carbohydrates, particularly Lewis Y, Tn, TF, SialylTn, GloboH, and gangliosides, such as GD2, GD3, GMl and GM2. Preferred epitopes are derived from antigens that are preferably expressed or over-expressed on epithelial tumours, such as breast cancer, cancer of the stomach and intestines, prostatic cancer, pancreatic cancer, ovarial cancer, cervical cancer, and lung cancer. Among the preferred epitopes are those which cause either a humoral immune response, i.e., induce a specific antibody formation in vivo

whereby not only antibodies of, for instance, the IgM class, but also antibodies of the IgG class will be formed in reaction to the administration of the multi-component preparation. Alternatively, also those antigens which generate T-cell-specific immune responses may, in particular, be selected as epitopes in the sense of the invention. Among those, also intracellular structures or T-cell peptides are to be selected as epitopes in the sense of the invention. Most preferably the immunogenic tumour-antigen-carrier conjugate preparation, or the multi- component vaccine, respectively, according to the invention is able to trigger a T-cell-specific immune response and a humoral immune response.

In a particular embodiment, at least two identical or different epitopes of an adhesion protein, for instance, a homophilic cellular membrane protein, such as EpCAM, are provided or mimicked on the antibody according to the invention. Thus, a plurality of antibodies specific for the same molecule, yet different EpCAM binding sites can be generated by active immunization.

The used multi-component tumour preparation according to the invention may, however, also be made available in the form of a glycosylated antibody, the glycosylation itself being also able to imitate an epitope of a carbohydrate epitope of a TAA.

In a particular embodiment, at least two different epitopes or epitope mimetics are provided, at least one epitope being derived from the group of peptides or proteins and at least one epitope being derived from the group of carbohydrates. An epitope of an EpCAM, CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, CA125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs protein and an epitope of a carbohydrate component, for instance of Lewis Y, Tn, TF, SialylTn, GloboH, and of glycolipids, particularly GD2, GD3, GMl and GM2 have turned out to be combined in a preferred manner. The epitopes can e.g. be coupled to the carrier by chemical means, by enzymatic reaction, or by molecular biological approach, such as recombination of the nucleic acids of epitopes and the carrier and their subsequent expression. This expression of one or more components can also be performed in a subject to be treated with the preparation.

A Lewis Y-glycosylated antibody having also a specificity for an EpCAM structure, in particular, constitutes an especially preferable immunogen in such a vaccine formulation. This antibody is particularly able to imitate cellular tumour antigens, and direct this immunogen

toward EpCAM expressing tumour cells, thereby inducing the desired immune response to inhibit epithelial tumour cells.

In a preferred manner, the immunogenic carrier according to the invention acts itself as an antigen, i.e. a protein antigen, in the vaccine, and additionally functions as the carrier for one or several additional epitopes, e.g. peptides or carbohydrate antigens. This means that tumour- antigen-carrier molecule according to the invention constitutes a multivalent antigen, for instance a bi-, tri- or polyvalent antigen. The epitopes are presented in a manner that cause the preparation to initiate an immune response against these epitopes. A preparation containing an antibody in the form of a di-, tri- or polyvalent antigen is thus provided.

In a preferred manner of the invention the carrier is highly immunogenic when applied to humans and monkeys. Preferably it is a xenogenic protein (i.e. protein derived from another species), an immunogenic protein or a heat-shock protein.

In a preferred embodiment of the invention the immunogenic carrier is an immunogenic antibody or a polyvalent antibody preparation, a xenogenic antibody, e.g. a murine antibody, or fragments of this antibody, which will exhibit a very strong immunogenic action in monkey or human organisms, which action can be further enhanced by a combination with adjuvants.

The tumour-antigen-carrrier conjugate according to the invention is primarily used for active immunization and, therefore, administered in small quantities only. Thus, no particular side- effects are to be expected even if the carrier according to the invention, e.g. an antibody, is derived from a non-human species such as, for instance, a murine antibody. It is, however, assumed that a recombinant, chimeric as well as a humanized or human antibody combined with xenogenic, i.e. immunogenic components, such as e.g. murine and human components are particularly safe for the administration in man. On the other hand, a murine portion contained in the antibody according to the invention is able to additionally provoke the immune response in man on account of its foreignness. Thus, in preferred embodiments the immunogenic carrier can be a human, humanized, chimeric, rabbit, rat or murine antibody or derivative or fragment thereof.

Although an antibody according to the invention may, of course, be derived from a native antibody optionally isolated from an organism or patient, an antibody derivative preferably selected from the group consisting of antibody fragments, conjugates or homologues, yet also complexes and adsorbates is usually employed. So in any case, whenever the term antibody within this disclosure is used, also derivatives, homologs or fragments thereof are included. In any event, it is preferred that the antibody derivative contains at least portions of the Fab fragment, preferably along with at least parts of the F(ab')2 fragment, and/or parts of the hinge region and/or of the Fc part of a lambda or kappa antibody.

Furthermore, a single-chain antibody derivative such as, for instance, a so-called single chain antibody may also be used as an epitope carrier in the context of the invention. The antibody according to the invention is preferably of the type of an immunoglobulin like IgG, IgM or IgA.

On the antibody according to the invention, other substances such as peptides, glycopeptides, carbohydrates, lipids or nucleic acids, yet also ionic groups such as phosphate groups, or even carrier molecules such as polyethylene glycol or KLH may be additionally contained in a covalent manner. These side groups themselves may possibly represent epitopes of TAAs in the sense of the present invention.

It is preferred according to the invention to use a monoclonal antibody which, as abl, comprises itself a specificity for a TAA so as to be possibly able to bind directly to a tumour cell or its derivative. This is to appropriately localize an immune response, optionally on the site of a tumour or disseminated tumour cell. The specificity of the antibody is preferably likewise selected from the above-mentioned groups of TAAs and, in particular, from the group consisting of EpCAM, CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, CA125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs protein, and Lewis Y, Tn, TF, SialylTn, GloboH, and of glycolipids, particularly GD2, GD3, GMl and GM2.

A particularly good immunogen for EpCAM is, for instance, an anti-EpCAM antibody that imitates or comprises at least one or at least two EpCAM epitopes, for instance by its EpCAM- similar idiotype.

In an alternative embodiment, the antibody used according to the invention may, however, also be selected so as to specifically bind an antibody. In the tumour vaccine according to the invention, especially anti-idiotypic antibodies, i.e. ab2, are preferably used for active immunization. These antibodies may be equipped with additional sequences or structures in order to obtain an immunogen according to the invention. In preferred embodiments the anti- idiotypic antibody mimics Lewis-Y or EpCAM.

Anti-idiotypical antibodies used according to the invention preferably recognize again the idiotype of an antibody directed against a TAA. Thus, an epitope of a TAA is already formed on the paratope of the anti-idiotypical antibody as mimicry for the TAA. The selection of the epitopes is again preferably made from the above-mentioned TAA groups. As an example, an anti-idiotypical antibody is used against glycan-specific antibodies, for instance, an anti-idiotypic antibody recognizing the idiotype of an anti-Lewis Y antibody, e.g. as described in EP 0 644 947.

The immunogenic antibody used according to the invention is, above all, suitable as a basis for pharmaceutical formulations and preparations and, in particular, vaccines. Preferred are pharmaceutical preparations containing pharmaceutically acceptable carriers. These include, for instance, adjuvants, buffers, salts, preservatives. These pharmaceutical preparations may, for instance, be used for the prophylaxis and therapy of cancer-associated pathological conditions such as the formation of metastases in cancer patients. To this end, antigen-presenting cells are specifically modulated in vivo or also ex vivo, in order to generate an immune response to the TAAs comprised by the immunogenic antibody.

A vaccine formulation preferred in accordance with the invention contains the immunogenic antibody only in small concentrations, for instance, in an immunogenic quantity ranging from 10 ng to 10 mg. Depending on the nature of the tumour-antigen-carrier conjugate, yet also on the auxiliary agents or adjuvants employed, the suitable immunogenic dose is selected to range approximately from 10 ng to 10 mg and, preferably, from 1 μg to 1 mg, even more preferably from 10 μg to 500 μg.

In an alternative embodiment a depot vaccine to be released to the organism over an extended period of time may, however, may also contain far larger antibody quantities such as, for

instance, at least 1 mg to more than 10 mg. The concentration is a function of the amount of liquid or suspended vaccine administered. A vaccine is usually provided in ready-to-use syringes having volumes of from 0.01 to 1 ml, preferably 0.1 to 0.75 ml.

The immunogenic multi-component anti-tumour preparation or vaccine according to the invention is preferably presented in a pharmaceutically acceptable carrier suitable for subcutaneous, intramuscular, but also intradermal or transdermal administration. Another mode of administration functions via the mucosal pathway, for instance, the vaccination by nasal or peroral administration. If solids are used as adjuvants, an adsorbate or a suspended mixture of the antibody with the adjuvants is, for instance, applied. In special embodiments, the vaccine is administered as a solution or a liquid vaccine contained in an aqueous solvent.

Vaccine units of the tumour vaccines are preferably provided in suitable ready-to-use syringes. Since an antibody is relatively stable as compared to TAAs, the vaccine according to the invention offers the essential advantage of being marketable as a storage-stable solution or suspension already in a ready-to-use form. A content of a preservative like thimerosal or any other preservative with improved tolerance is not necessarily required, yet may be provided in the formulation to extend storage life at storage temperatures from refrigerator temperature to room temperature. The vaccine according to the invention may, however, also be provided in frozen or lyophilized form to be thawed or reconstituted on demand.

In any event, it has proven successful to enhance the immunogenicity of the multi-component vaccine according to the invention by the use of adjuvants. To this end, the vaccine is co- formulated or co-applied with adjuvants such as, for instance, aluminum hydroxide (AIu gel) or phosphate, e.g. growth factors, lymphokines, cytokines such as TNF-alpha, TNF-beta, IL-I -beta, IL-2, IL-4, IL-6, IL-7, IL-8, IL- 12, IL- 18, IL-21, IL-23, GM-CSF, (IFN-gamma) gamma interferon, IFN-alpha, IFN-beta, IFN-omega, or factors of the complement system, such as C3d and, furthermore, liposome preparations, lipopolysaccharide (LPS) from E. coli or derivatives of LPS. Yet, also formulations with additional antigens against which the immune system has already induced strong immune responses, such as tetanus toxoid, bacterial toxins like Pseudomonas exotoxins, Diphteria toxin, and derivatives of lipid A are envisaged. In a preferred embodiment of the invention adjuvant semi-synthetic adjuvants, such as QS-21, or ENHANZYN or related adjuvants derived from natural sources, or alternatively cationic or anionic polymers,

such as polylysine, polyarginine, polyethylenimine (PEI), or derived products such as IC31 are co-formulated with the vaccine. Further adjuvants are preferably selected from one or more of the group of tetanus toxoid, bacterial toxins, preferably Pseudomonas exotoxins, Diphteria toxin, derivatives of lipid A. As examples of additional antigens, against which the immune system has already induced strong immune responses.

In an alternative embodiment of the invention water-in-oil, oil-in-oil, or oil-in water formulations are included in the multi-component vaccine to increase immunogenicity and/or serve as a depot for sustained release of immunogen and/or adjuvants.

In a particularly preferred embodiment of the invention nanoparticular structures ("nanoparticle") or microparticular structures ("microparticles") or other particulate slow-release formulations, e.g. heat-treated aluminium hydroxide formulations or co-formulated aluminium hydroxide particles, are used for preparation of the said multi-component vaccine. The inventive slow-release formulation can be achieved by a co-formulation of the components (antigen/carrier/adjuvant) with such a slow-release mediator, in particular in particulate form. A preferred slow-release formulation is achieved by co-formulation with aluminium hydroxide in particulate form which coadsorbs the components. Aluminium hydroxide is generally also a weak adjuvant. However, for the purposes of the present invention, it is primarily used for slow- release mediation by co-adsorbing the components of the preparation. As adjuvant preferably strong adjuvans, like QS-21, are used. Since aluminium hydroxide is only a weak adjuvant and may preferably be used as slow-release mediator the adjuvant (iii) is a different compound than the slow-release mediator. The strength of the adjuvant is potentiated by adsorption onto a particulate structure provided by the aluminium hydroxide. Importantly, both the protein carrier with covalently coupled antigen epitopes and the strong adjuvant are physically localized together onto the particulate structure provided by aluminium hydroxide or other substances such as aluminium phosphate, calcium phosphate etc.. The slow-release formulation - in order to coadsorb the components - should not be formulated in the presence of substances interfering with the adsorption onto/into the particulate formulation. Such interfering substances are e.g. blood proteins. However, after a stable slow-release formulation has been obtained it can provide also slow release characteristics even after application of the formulation into an environment containing biological fluids, e.g. blood, serum, tissue fluid, interstitial fluid. In a preferred embodiment the slow-release formulation comprises particulate structures, preferably formed by

adsorption onto the particulate structure, in particular preferred aluminium hydroxide, aluminium phosphate or calcium phosphate. The nanoparticles, microparticles or other particular slow- release formulations are used to increase the immunogenicity of the vaccine formulation by enhancing uptake of antigen in antigen presenting cells (APC) and/or enabling a sustained release of antigen/immunogen and/or adjuvants. Preferred embodiments, the slow-release formulation desorbes the epitopes and/or the carrier by at least 50%, preferably by at least 80%, more preferred by at least 90%, in particular preferred by at least 99%, slower than the preparation without slow-release formulation, e.g. without the nano- or microparticulate structures.

To formulate vaccines, also other known methods for conjugating or denaturizing vaccine components may be employed in order to further enhance the immunogenity of the active substance.

Particular embodiments of the vaccine according to the invention contain additional vaccination antigens, particularly anti-idiotypical antibodies, i.e., mixtures of the immunogenic tumour- antigen carrier conjugates according to the invention with several of them administered at the same time.

The immunogenic antibody according to the invention is also suitable for the preparation of diagnostic agents according to the invention. Thus, reagents containing the immunogenic antibody in association with other reactants or detection agents may be offered as diagnostic agents in set form. Such an agent preferably contains a label for the immediate detection of the antibody or its reaction product. The diagnostic agent according to the invention is, for instance, used for the qualitative and/or quantitative assessment of tumour cells or metastases or the determination of a metastasizing potential, said agent acting by an immune reaction or immune complexation.

The multi-component preparation useful for the present invention can be produced by a method comprising the steps of: a) providing a carrier molecule, e.g. immunogenic protein, e.g. an antibody; b) coupling at least two epitopes of a tumour-associated antigen to said carrier molecule; and c) formulating the coupling product from step b) into a slow-release formulation.

Alternatively, the method according to the invention may already depart from an anti-idiotypical antibody, the method steps in that case comprising: a) providing an antibody including the idiotype of a tumour-associated antigen (e.g. anti- idiotypic antibody mimicking a TAA); b) coupling at least one epitope of a tumour-associated antigen to said antibody; and c) formulating the coupling product from step b) into a slow-release formulation.

Coupling is usually performed by chemical or biological, e.g. enzymatic, reactions. The connection of an antibody with an epitope is, however, also feasible already on a molecular biological level. A conjugated product can be expressed and prepared just by the recombination of nucleic acids. Such methods according to the invention are characterized by the steps of: a) providing a nucleic acid encoding an antibody including the idiotype of a tumour-associated antigen; and b) recombining said nucleic acid with a nucleic acid encoding an epitope of a tumour-associated antigen or its mimicry; or a) providing a nucleic acid encoding an antibody; b) recombining said nucleic acid with one or several nucleic acid(s) encoding at least two epitopes of a tumour-associated antigen or its mimicry; and c) formulating the coupling product from step b) into a slow-release formulation.

The slow-release formulation can be created by e.g. the addition and co-formulation of nano- or microparticular structures, e.g. with a high surface area, leading to adsorption of the components of the preparation or by heat treatment of the epitope/carrier together with an adjuvant as described in the US 2003/0143221 Al. Thus, the step of formulating the coupled products into the slow-release formulation comprises in this embodiments the heat adhesion or particulate adsorption onto or into a slow-release mediator, preferably aluminium hydroxide or calcium phosphate, or the integration into a liposome, polymeric microspheres, dextran, cationic lipids or nano- or microparticles.

Preferably, the slow-release formulation is co-formulated with an adjuvant as mentioned above. In particular preferred embodiments formulating the coupled products into the slow-release formulation comprises co-formulating the coupled products with an adjuvant, preferably growth

factors, lymphokines, cytokines such as TNF-alpha, TNF-beta, IL-I beta, IL-2, IL-4, IL-6, IL-7, IL-8, IL- 12, IL-18, IL-21, IL-23, GM-CSF, IFN-gamma, IFN-alpha, IFN-beta, IFN-omega, or factors of the complement system, such as C3d, liposome preparations, lipopolysaccharide, LPS derivatives, lipid A, semisynthetic adjuvants, in particular QS-21 or ENHANZYN, adjuvants derived from natural sources, cationic or anionic polymers, such as polylysine, polyarginine, polyethylenimine (PEI), or IC31, and aluminium hydroxide and/or calcium phosphate, nano- or microparticles, liposomes, polymeric microspheres, dextran or cationic lipids resulting in co- adsorption or co-integration.

Alternatively, the carrier, on which the invention is based, may, for instance, be an anti-idiotypic antibody, i.e. an ab2, and/or an antibody having the specificity for at least one epitope of at least one tumour-associated antigen, i.e. an abl.

During the coupling procedure the two molecules are conjugated via the formation of a covalent bond. A derivative that differs from native antibodies will, thus, be synthesized. The combination according to the invention, of two immunogenic TAA mimicries completely different in nature in a surprising manner allows for an extremely efficient immunization against tumour-associated or tumour-specific structures such that the endogenous immune system will be efficiently protected against the respective tumours or able to combat these tumours.

The antibody according to the invention functions as a protein antigen-carrier which is present, for instance, with a carbohydrate antigen to constitute a conjugate of the invention. It is likewise feasible to provide several carbohydrate antigens in the conjugate according to the invention. Thus, several different glycans triggering immune responses against two or several different tumour-associated carbohydrate structures may, for instance, be coupled to one antibody. Such a conjugate does not occur in nature. The auto-antigenic structures are thereby recognized as foreign, which will additionally increase immunogenity. In accordance with the invention, a conjugate of this type is, therefore, present in a synthetic composition naturally occurring neither sterically nor functionally (i.e., in tumour cells).

According to the invention, the coupling of two structures completely different in nature to a carrier molecule - in addition to the advantage of a simple formulation of the synthetic vaccine - also results in a much simpler vaccination scheme because the same vaccine can be given during both, the initial vaccination and the subsequent booster vaccinations.

Moreover, the invention relates to a method for "immunogenizing" epitopes of TAAs or their mimicries. To this end, primarily low-molecular epitopes of the antigens are used, which by themselves would hardly be recognized by the immune system of mammals, particularly man. Immunogenization is affected in a manner that an antigen is conjugated to an antibody, with the antibody functioning as a carrier. By the method according to the invention, it is feasible to render immunogenic a plurality of epitopes and naturally, in particular, the epitopes of the already mentioned selection of antigens. The immunogenic antibody produced according to the invention preferably contains the epitope to be immunogenized and a further epitope of a tumour-associated antigen.

Immunogenization yields a material that is surprisingly well apt for the immunization of patients. The product to be obtained by the invention is, therefore, preferably provided as a vaccine.

Methods for detecting suitable antigenic structures, modelling and preparing TAA-derived peptides, polypeptides or proteins, or nucleic acids encoding the same, and, furthermore, lipoproteins, glycolipids, carbohydrates or lipids are known to the skilled artisan and can be provided for the respective tumour-specific structure without much experimental expenditure. Furthermore, methods for conjugating proteins with such structures are known, which are suitable for the method according to the invention.

The carbohydrate structures selected as epitope mimicry can be derived from natural or synthetic sources, the carbohydrates being present as glycoproteins or glycolipids and capable of being coupled as such to the respective carrier molecule.

Also the antibody components can be chemically synthesized and subsequently connected with epitope structures, or synthesized together. Upon the chemical synthesis of antibody carrier molecules, it is feasible to introduce reactive groups on particular sites in order to be able to control both the extent of coupling with an epitope and the type and location of the bond.

The antibody carriers can also be produced as recombinant molecules by genetic engineering. It is conceivable to produce these antibodies in host cells that do not affect glycosylation (such as,

e.g., Escherichia coli). Such polypeptides may then be chemically or enzymatically coupled to a desired carbohydrate antigen.

It is, however, also conceivable that the antibody carrier is produced in cells that are able to perform glycosylation of the molecule. The genetic modification of nucleic acids encoding native antibodies may, for instance, cause the formation of appropriate glycosylation sites in the translated molecule.

The glycosylation of such a recombinant gene product with the respective rumour-associated glycan structures can be affected by production in cells genetically modified to appropriately glycosylate proteins. Such cells may be natural isolates (cell clones) than can be found by adequate screening for the desired glycosylation.

It is, however, also feasible to modify cells in a manner that they will express the respective enzymes necessary for the desired glycosylation, such that the desired glycosylation on the recombinant polypeptide carrier protein will be exactly found (Glycoconj. J. (1999), 16:81). It is, however, also feasible to enzymatically produce, or modify, the glycosylation patterns of proteins (Clin. Chem. Lab. Med. (1998), 36:373).

In the tumour-antigen-carrier conjugate according to the invention, the various epitope structures may be coupled to one another via a linker molecule. Such a linker is preferably comprised of a short, bifunctional molecule such as, e.g., N-hydroxysuccinimide. Coupling of nitrophenyl- activated carbohydrates to primary amino groups of the carrier is also a preferred method of coupling according to the invention. In a further preferred embodiment, coupling is done via sulfhydryl groups (Biochim. Biophys. Acta (1983), 761, 152-162). Examples of sulfhydryl- reactive linkers are BMH, DFDNB or DPDPB. Yet, the reactive linker may also be realized by a larger chemical compound than a simple coupler molecule. The prerequisite always being that such a coupler will not adversely affect the immunological properties of the conjugate, i.e., will not itself trigger any substantial immunogenity. According to the invention, a coupler may also be produced quasi- "in situ" by the chemical conversion of a portion of the antibody or the structure to be conjugated. This coupler produced on the antibody or epitope structure itself can then be directly conjugated to the respectively other binding partner (e.g., via the amine group of lysine, via OH groups, sulfur groups, etc.). Coupling methods are known from the prior art

(Anal. Biochem, (1986) 156, 220-222; Proc. Natl. Acad. ScL, (1981), 78, 2086-2089; Biochem. Biophys. Res. Comm. (1983), 115, 29-37).

According to a particular embodiment of the present invention, the antibody according to the invention comprises a nucleic acid molecule encoding a proteinic TAA as an epitope structure in the sense of the present invention, said nucleic acid being covalently conjugated.

The present invention also relates to a set suitable for tumour vaccination. The set comprises a preparation of a multi-component vaccine according to the invention and a suitable application means such as, e.g., syringes, infusion devices, etc. If the conjugate preparation is present in lyophilized form, the set will further comprise a suitable reconstitution solution optionally including special stabilizers or reconstitution accelerators.

The present invention, by which the immunogenic carrier coupled with several different epitope structures and, in particular, the structure of a tumour-associated carbohydrate antigen is provided, enables the triggering of an immune response having two or more specificities and, thus, combating a tumour cell by two or more different tumour-associated antigens. As a result, the effective range of the vaccine is widened and more specifically designed.

The invention will be explained in more detail by way of the following examples and the examples provided and the figures of the drawing, yet without being limited thereto.

FIG. 1 illustrates Carbohydrate and mucin antigens as attractive targets for cancer vaccination. Carbohydrate antigens formed by aberrant glycosylation and present on the surface of tumour cells are prominent tumour-associated antigens (TAAs) and represent promising targets for anticancer immunization.

FIG. 2 shows that targeting multiple antigens is an essential pre-requisite for therapeutic efficacy due to heterogenicity in TAA expression, illustrated on the example of the sub-indications of breast cancer.

FIG. 3 depicts the coupling chemistry: Linkage of nitrophenyl-activated ligands, such as synthetic oligosaccharides or peptides with a spacer linker (sp), to highly immunogenic protein

carriers by one standardized coupling procedure, e.g. coupling to the primary amino groups of the carrier protein.

FIG. 4 illustrates the coupling of a SialylTn Carbohydrate epitope to a protein carrier: Coupling Chemistry: Linkage of nitrophenyl-activated synthetic spacered SialylTn to the primary amino groups of the protein carrier. Formulation of multicomponent Tumour vaccine: Formulation (Adsorption) onto aluminium hydroxide and co-formulation (co-adsorption) of additional strong adjuvant(s).

FIG. 5 shows the characterization of the SialylTn-mAbl7-lA coupling product by LDS-PAGE (A), Western Blots (B), and IEF (Iso-Electrical Focusing, C) and Uptake (D) of vaccine formulation particles (red) in adherent monocytes (nuclei of monocytes shown in blue).

FIG. 6 shows the data on the immune responses against carrier protein and SialylTn after immunization with SialylTn-mAbl7-lA vaccine with or without co-formulation with QS-21.

FIG. 7 shows the specificity of immune responses against SialylTn and mAbl7-lA carrier.

FIG. 8 shows the cell binding of induced reactivity against SialylTn positive OVCAR-3 tumour cells (FACS analysis).

FIG. 9 shows ADCC (Antibody- Dependent Cellular Cytotoxicity) of induced immune response induced in immunized animals against SialylTn expressing OVCAR-3 tumour cells.

FIG. 10 shows cell binding of induced immune response to (SialylTn negative) KATOIII tumour cells.

FIG. 11 shows the measured cytokine release after immunization with SialylTn-mAbl7-lA vaccine with or without QS-21 adjuvant.

FIG. 12 shows the kinetics of immune response against SialylTn coinciding with a temporary cytokine release in serum.

FIG. 13 shows NK cell mediated lysis of KATOIII tumour cells after in vitro activation of human NK cells by immune serum of immunized Rhesus monkeys.

FIG. 14 shows lysis of KATOIII tumour cells by Rhesus monkey derived PBMCs after repeated boost immunizations.

FIG 15 shows the determination of efficacy of coupling of a Lewis- Y carbohydrate to an EpCAM-specific antibody: Western blot of coupling product vs. uncoupled carrier.

FIG 16 illustrates the concept of anti-idiotypic antibodies, that mimic tumour associated antigens (carbohydrates or peptides) as immunogenic carrier (graphical depiction of the immunolgical concept. Symbols: Blue = Lewis-Y mimic, Red = coupled Sialyl-Tn, Green = coupled EpCAM derived epitope).

Examples

Example 1

Carbohydrate and mucin antigens are attractive targets for cancer vaccination

There is a growing body of data from preclinical and clinical studies demonstrating that actively induced antibodies or immune effector cells may have a deciding role in cancer treatment of tumour cell. Elimination of circulating tumour cells and micro-metastases or eradication of tumour cells remaining after surgery or radiotherapy are the major targets of cancer immunotherapy.

The choice of the appropriate target antigen(s), so called tumour associated antigen(s) (TAA), is essential for the efficacy of immune therapy. Carbohydrate and mucin antigens resulting from aberrant glycosylation in tumour cells are present on the surface of tumour cells. They can serve as susceptible targets for immune therapy in experimental animals and cancer patients.

Fig. 1

SialylTn is a truncated o-glycosylated short carbohydrate resulting from abberant glycosylation of the mucin as typical for cancer cells (right side) as compared to a more complex glycosylation of the mucin present on normal cells (left side)

SialylTn is expressed in more than 80% of cancers of breast, colorectal, prostate, uterus and ovarian origin with no or very limited expression on the corresponding normal tissues. SialylTn expression by various epithelial cancers correlates with a more aggressive phenotype and poorer prognosis.

Example 2

Targeting multiple antigens is an essential pre-requisite for therapeutic efficacy

The heterogeneous expression of TAAs on cancer cells, poses the need to target more than one antigen for maximal eradication of tumour cells and to minimize the threat of escape variants.

Furthermore, combining several immunological principles on a single molecule would be pharmaceutically most favorable.

Fig. 2

Heterogenicity in TAA expression is illustrated in this figure showing the incidence of LewisY and EpCAM expression in different sub-indications of breast cancer. In order to achieve a therapeutic effect in > 50% of tumours of a given tumour indication a single target anti LewisY vaccine will show sufficient efficacy only in two (minor) indications (tubular cancer and papillary cancer), representing only < 5% of overall breast cancer. Respectively, a single target anti EpCAM vaccine will show efficacy in 5 minor indications representing < 10% of breast cancer.

A bivalent vaccine ("Multi-epitope vaccine") targeting both LewisY and EpCAM will show efficacy in most breast cancer indications (representing > 75% of breast cancer). This example illustrates the power of vaccines that target more than one tumour antigen epitope.

Example 3

Multi-epitope Tumour-antigen-conjugate vaccine

Coupling of tumour-associated carbohydrate epitopes is done to immunogenic protein carriers, e.g. murine antibody, resulting in increased immunogenicity of the carbohydrate antigen and provides T cell help.

In an optimal setting the carrier molecule itself also induces anti-tumour reactivity (e.g. MMA383 and mAbl7-lA) adding the strong anti-carrier immune response to the immune response against the carbohydrate TAAs. In any case, the coupling of more than one carbohydrate antigen to the immunogenic carrier will generate a multi-epitope vaccine.

According to the present invention a variety of carrier molecules are suitable: While the primary intention of this technology is to increase the immunogenicity of the per se non-immunogenic carbohydrate antigens, this technology also offers the potential to make use of additional immunogenic principle provided by the carrier molecule.

• the carrier molecule itself can mediate a meaningful immune response against tumour associated epitopes, e.g. using an anti-idiotypic antibodies, for example: MMA383 which is an anti-idiotypic antibody for LeY

• the carrier molecule can target cell surface structures resulting in additional immune stimulation (e.g. Diphteria toxin, Anti-TLR9 Ab: antibody targeting the toll like receptor 9 (TLR9) present on antigen presenting cells (APCs) with potential intrinsic adjuvant effect)

According to the present invention the following protein carrier providing themselves epitope for tumour-associated antigens are preferred: including EpCAM, CEA, TAG-72, MUCl, EGF, EGF-R, folate binding protein A-33, CA125, EpCAM, HER-2/neu, PSA, MART, MAGEs, GAGEs, BAGEs proteins.

According to the present invention epitopes of the following antigens are preferably coupled to the carrier protein: Lewis Y, Tn, TF, SialylTn, GloboH, and GD2, GD3, GMl and GM2

Fig. 3

Coupling chemistry: Linkage of nitrophenyl-activated ligands, such as synthetic oligosaccharides, chemical entities) or peptides with a spacer linker (sp), to highly immunogenic protein carriers by one standardized coupling procedure, e.g. coupling to the primary amino groups of the carrier protein.

Example 4

Coupling of SialylTn Carbohydrate to an EpCAM-Specific Antibody (mAbl7-lA) and Formulation of the multi-component vaccine. Co-Formulation of additional adjuvants on anluminium hydroxide.

Methods:

Coupling of SialylTn carbohydrate to mAbl7-lA

The SialylTn carbohydrate antigen was coupled to the mAbl7-lA (murine IgG2a) protein carrier at a molar ratio of SialylTn to mAbl7-l A of 18:1 by reacting 10 mg of nitrophenylated spacered SialylTn, Neu5Acα2-6GalNAcα-O(CH2)3NHCO(CH ₂ )4COO-(p-NO ₂ C6H4) (MW 819 g/mol, Lectinity, Finland), with lOOmg of mAbl7-lA. Briefly, 100 mg of mAbl7-lA (10mg/ml) were dialyzed twice at 4°C for 20 h against 700 ml Coupling Buffer (0.1 M Na-PO ₄ , 0.15 M NaCl, pH 8.5) using a Slide-A-Lyzer dialysis cassette MWCO 1OK (Pierce). Concentration of mAbl7-lA was determined by Size Exclusion Chromatography (SEC). In parallel, 10 mg of Neu5Acα2- 6GalNAcψ-O(CH ₂ )3NHCO(CH2)4COO-(p-Nθ2C ₆ H4) were dissolved in 300 μl DMF and added to the ice cold mAbl7-lA. The reaction mixture was incubated rotating at +4°C. The kinetic of the reaction was monitored by the size of the coupling product as analyzed by SEC. After 28 h the reaction mixture was dialyzed against Formulation Buffer (1 mM Na-PO ₄ , 0.86% NaCl, pH 6) using Slide-A-Lyzer dialysis Cassette 3.5K (Pierce) at +4°C for 20 h. For comparison, uncoupled mAbl7-lA was processed in parallel. SialylTn-mAbl7-lA coupling products were analyzed by size exclusion chromatography, LDS-PAGE, Western blot, isoelectric focusing (IEF), and Resorcinol assay.

Analysis of coupling products (tumour-antigen-carrier conjugates)

Size Exclusion Chromatography

Concentrations of SialylTn-mAbl7-lA coupling products were quantified by size exclusion chromatography (SEC) on a ZORBAX GF-250 column in a Dionex system and on a TSKgel G3000SW column in a HPl 100 system.

IEF, LDS-PAGE and Western Blots

SialylTn-mAbl7-lA coupling products were analyzed by SERVALYT PRECOTES horizontal flat bed IEF electrophoresis pH 3-10 (SERVA) followed by Coomassie blue staining (Invitrogen), and lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE,

NuPAGE Electrophoresis System, Bis-Tris-Gel, 4-12%) under reducing conditions (DTT, 1,4- Dithiothretiol) followed by SilverXpress™-staining (Invitrogen).

For Western blot analysis, samples following LDS-PAGE were transferred (25V, 1.1 W, 1.5h) to Immobilon membranes (PVDF 0,45 μm, Millipore). Membranes were blocked with 3% skim milk and stained with rabbit anti-mouse IgG (H+L)-HRP (1:1000, Zymed) or alternatively with anti-SialylTn CD175s (IgGl) (lOμg/ml, DAKO) and rat anti-mouse IgGl-HRP (1:1000, Becton Dickinson).

Quantification of Sialic acid by Resorcinol assay

Amount of sialic acid in the coupling products was quantified by Resorcinol-HCl reaction in the presence of CuSO4, and spectrophotometric measurement of absorbance at OD ₅ 62. Briefly, 0.15ml of samples and 0.15ml Resorcinol reagent (2% Resorcinol, 2.5mM CuSO ₄ , HCL (37%)) were mixed in tightly closed glass vials, and incubated at 100°C for Ih. The color reaction was measured by adsorption at OD ₅ 62 (Spectrophotometer Ultrospec II, LKB Biochrom). As a standard a 'SialylTn mix' (consisting of Sialic acid (i.e. N-Acetylneuraminic acid) and GaINAc at a molar ratio 1:1), 1.25 - 25 μg/ml was used for calibration.

Multi-component vaccine Formulation

500 μg of SialylTn-mAbl7-lA conjugate were adsorbed on 1.67 mg aluminum hydroxide in 0.5 ml formulation buffer (ImM Na-PO ₄ , 0.86% NaCl, pH6). Vaccine was formulated either w/o additional adjuvant or co-formulated and thus co-adsorbed with lOOμg QS-21 adjuvant (Antigenics Inc., Lexington, MA).

LAL Assay

Amounts of endotoxin in the vaccine formulations were estimated by Limulus Amebocyte Lysate - Endochrome™ assay (Charles River Endosafe) according to the manufacturer. None of the final vaccine formulations contained detectable amounts of endotoxin.

Pyrogenicity test in rabbits

Final vaccine formulations were subjected to pyrogenicity test by i.v. application in rabbits. The formulations used for this study were negative regarding pyrogenicity testing with the sum of individual temperature rise recorded in three rabbits being +0 ⁰ C (w/o additional adjuvant) and +0.3°C for vaccines co-formulated with QS-21 adjuvant.

Multicomponent vaccine formulation with Alexa Fluor 546 labelled SialylTN-mAbl7-lA conjugate

1 mg of SialylTn-mAbl7-l A conjugate was labelled with Alexa Fluor 546 - a fluorescence dye, according to the Instruction Booklet of the Alexa Fluor 546 protein labelling Kit (A- 10237 from

Molecular Probes, USA).

500 μg of SialylTn-mAbl7-lA conjugate were adsorbed on 1.67 mg aluminium hydroxide in 0.5 ml formulation buffer (ImM Na-PO ₄ , 0.86% NaCl, pH6).

Incubation of multicomponent vaccine formulation with Alexa Fluor 546 labelled SialylTn- mAbl7-lA conjugate with human monocytes from peripheral blood.

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors (obtained from the Austrian Red Cross) on a Ficoll density gradient. The monocyte fraction was obtained by 1 h plastic adherence. Adherent monocytes, cultured in RPMI and 10% FCS, were incubated with 10 microliter the aluminium hydroxide-adsorbed Alexa Fluor 546 labelled SialylTn-mAbl7-lA conjugate formulation for 1 hour. The adherent monocytes were washed three times with PBS and incubated with PBS with the DNA staining fluorescence dye DAPI, washed another three times and investigated under a Axioplan Fluorescence microscope.

Results:

Nitrophenyl-activated, spacered SialylTn was reacted with mAbl7-lA at a molar ratio of 18:1. The kinetic of the reaction was monitored by SEC, and the reaction was stopped when no further increase in the size of the coupling product was detected. The SialylTn-mAbl7-lA coupling product was analyzed by LDS-PAGE under reducing conditions followed by Silver staining (Fig. 5A) and Western blotting stained with anti-SialylTn CD175s (IgGl)) and rat anti-mouse IgGl- HRP (Fig. 5B). LDS-PAGE showed an increase in the molecular weight of the heavy chains (5OkDa) and the light chains (25kDa) in the coupling products (A: lanes 3,5,7,9 compared to uncoupled mAbl7-lA, lanes 2,4,6,8) indicating that SialylTn has been coupled to both, heavy and light chains of the mAbl7-lA. Western blot analysis showed that SialylTn was detectable in the SialylTn-mAbl7-lA coupling products but not in the mAbl7-lA (Fig. 5B, lane 2,4,6 vs. lane 1,3,5 (uncoupled mAbl7-lA). Coupling of the (negatively charged) SialylTn to mAbl7-lA resulted in a shift of the isoelectric point as shown by IEF (Fig. 5C, lane 2: SialylTn-mAbl7-lA,

lane 3: mAbl7-lA). The molar ratio of SialylTn to mAbl7-lA in the coupling product was quantified using the Resorcinol assay. In this assay the amount of sialic acid in the coupling products is quantified by Resorcinol-HCl reaction in the presence of CuSθ4. A molar ratio of SialylTn to mAbl7-lA in the coupling product of approximately 14:1 was determined by Resorcinol assay.

The SialylTn-mAbl7-lA coupling product (500μg) was adsorbed on aluminum hydroxide, aliquoted into sterile vials. The aluminium adsorbed multi-component vaccine was used either w/o additional adjuvant or in a co-formulation with lOOμg QS-21 adjuvant. The vaccine formulations were analyzed for traces of endotoxin by Limulus Amebocyte Lysate and pyrogenicity test by i.v. application in rabbits. Neither elevated endotoxin levels nor significant pyrogenicity were detected.

Following incubation with aluminium hydroxide-adsorbed Alexa Fluor 546 labelled SialylTn- mAbl7-l A conjugate formulation adherent monocytes were investigated under a Axioplan Fluorescence microscope. As shown in Fig. 5D, the nuclei of the adherent monocytes are stained blue by the DNA dye DAPI. In the cytoplasm of the monocytes multiple particulate inclusions with the typical red fluorescence from Alexa Fluor 546 can be seen, indicating a dramatic uptake of vaccine formulation into the monocytes, i.e. antigen-presenting cells.

Fig. 4

Coupling of a SialylTn Carbohydrate epitope to a protein carrier:

Linkage of nitrophenyl-activated synthetic spacered SialylTn to the primary amino groups of the protein carrier (e.g. murine monoclonal EpCAM binding antibody, mAbl7-lA).

Fig. 5 Characterization of the SialylTn-mAbl7-lA coupling product by LDS-PAGE, Western Blots, and IEF, and Illustriation of uptake of Multicomponent tumorvaccine into antigen presenting cells, such as blood derived monocytes

Unconjugated mAbl7-lA and SialylTn-mAbl7-lA were subjected to LDS-PAGE under reducing conditions followed by silver staining (A: mAbl7-lA (lane 2,4,6,8) and SialylTn- mAbl7-lA (lane 3,5,7,9), at two serial dilutions, respectively) (markers 1 and 10), or blotted and stained with anti-SialylTn CD175s (mlgGl) and rat anti-mouse IgGl-HRP (B: mAbl7-lA (lane 1,3,5,7) and SialylTn-mAbl7-lA (lane 2,4,6,8), positive control: lane 9; marker lane 10).

Heavy (5OkDa) and light (25kDa) chains of mAbl7-lA or conjugated SialylTn-mAbl7-lA under reducing conditions can be seen (A, B).

Non-conjugated mAbl7-lA and SialylTn-mAbl7-lA were also subjected to IEF-PAGE and stained by Coomassie blue (C: SialylTn-mAbl7-lA (lane 2) and mAbl7-lA (lane 3), marker

(lanes 1, 4, 5)).

D: Incubation of multicomponent vaccine formulation with Alexa Fluor 546 labelled SialylTn- mAbl7-lA conjugate with human monocytes from peripheral blood. The nuclei of the monocytes are stained blue by the DNA binding dye DAPI. In the cytoplasm of the monocytes multiple particulate inclusions with the typical red fluorescence from Alexa Fluor 546 can be seen, indicating a dramatic uptake of vaccine formulation into the monocytes.

Example 5:

Immunization of Rhesus monkeys with SialylTn-mAbl7-lA multi-component vaccine and analysis of the induced immune responses against carrier and coupled tumour-antigen (SialylTn): Effect of additional adjuvant (QS-21)

Methods:

Rhesus monkey immunization study

Safety, tolerability and immunogenicity of multiple subcutaneous injections of IGN402 were evaluated in vaccination studies in Rhesus monkeys. All animal studies were performed under controlled and documented conditions in accordance with animal health care standards at Biotest Ltd., Konarovice, Czech Republic. Per group, four healthy adult Rhesus monkeys (age and sex matched, group I: #152, #258, #292, #330 and group II: #38, #269, #308, #382, without or with QS-21, respectively) were vaccinated on days (d) 1, 15, 29 and 57 by subcutaneous injection and re-boosted on d226. Blood samples were taken before (d -7 and d -3) and after immunization (days 15, 22, 29, 43, 57, 71, 85, 99) and d226 (before re-boost) and d240 (two weeks after re- boost) for serum analytic.

In a second Rhesus monkey study animals - four animals per group (#31, #48, #76, #318) - were vaccinated with four initial immunizations of IGN402 co-formulated with QS-21 on days (d) 1, 21, 49 and 76. Heparinized blood samples were taken before (d 1) and after immunization (d38, 60, and 86) for preparation of PBMC.

All immunizations were well tolerated by all animals with no signs of systemic or local toxicity related to immunization.

ELISA for immune reactivity against mAbl7-lA

Pre-sera and immune sera of Rhesus monkeys, i.e. sera obtained before and after immunization, respectively, were analyzed regarding the induced immune response against mAbl7-lA by ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated with 10 μg/ml mAbl7-lA. Wells were blocked with 10 % FCS in PBS for Ih at 37 ⁰ C and samples pre-diluted in PBS with 2 % FCS were incubated for 1.5 h at 37°C. A positive control serum (derived from a Rhesus monkey immunized with mAbl7-lA formulated on alhydrogel) with known reactivity against mAbl7-lA was tested in parallel and used for normalization of OD values between different ELISA plates. For detection plates were incubated with a 1 :2000 diluted sheep anti-human IgG- (γ-chain)-HRP conjugate (Chemicon) for 30 min at 37°C. Staining with substrate OPD (10 mg OPD dissolved in 25 ml staining buffer containing 10 μl 30 % H2O2) was stopped by adding 50 μl H2SO4 (30 %) and the color intensity was measured at OD492/620. The titer was defined as reciprocal serum dilution yielding an absorbance of OD = 1.0 on a titration curve. Curve fitting was done using GraphPad Prism version 4.0.

SialylTn-PAA ELISA

Pre-serum and immune sera of Rhesus monkeys were tested for reactivity against the synthetic SialylTn carbohydrate antigen by SialylTn-PAA ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated lOμg/ml SialylTn-PAA (Lectinity, Finland). Wells were blocked with PBS containing 10 % FCS for 1 h at 37°C. Serum samples were pre-diluted in PBS containing 2 % FCS and 5 % glucose, and incubated for 2 h at 37 ⁰ C. A positive control serum with known reactivity against SialylTn was used for normalization between different ELISA plates. For detection plates were incubated with 1:2000 diluted mouse anti-human IgM-HRP conjugate (Southern Biotechnology) or 1:2000 diluted sheep anti-human IgG-(γ-chain)-HRP conjugate (Chemicon), respectively, for 30 min at 37 ⁰ C. Staining with substrate OPD (lOmg OPD dissolved in 25 ml staining buffer containing 10 μl 30 % H ₂ O ₂ ) was stopped by adding 50 μl H2SO4 (30 %) and the color intensity was measured at OD492/620. Titers were defined as reciprocal of serum dilutions yielding an absorbance of OD = LO and OD = 0.5 for IgM and IgG, respectively. Curve fitting was done using GraphPad Prism version 4.0.

Results:

Immunization of Rhesus monkeys with SialylTn-mAbl7-lA in the presence or absence of QS- 21 adjuvant

SialylTn-mAbl7-lA conjugate was formulated onto alhydrogel, either with or without QS-21 adjuvant. Application of both vaccine formulations to Rhesus monkeys was well tolerated by all animals. The immune responses against the SialylTn carbohydrate antigen (IgG, IgM) and the mAbl7-lA carrier protein (IgG) were analyzed by respective ELISA. The summarized data of the anti-SialylTn immune response and the immune response against the mAbl7-lA carrier molecule are shown in Fig. 6. Both vaccines induced high IgG titers against the carrier molecule. In the presence of QS-21 the immune response against the carrier showed a slightly more rapid onset (significant titers at day 15 with QS-21 vs. day 22 without QS-21) and approximately 5-10 fold higher peak titers as compared to the vaccine without QS-21. A similar kinetic was found for the IgM response against SialylTn, with a more rapid onset and approximately 10-fold higher IgM titers in the presence of QS-21 adjuvant. The most dramatic effect of QS-21 adjuvant was the induction of a pronounced SialylTn-specific IgG response in the QS-21 adjuvanted group. In three out of four animals a SialylTn-specific IgG response was measurable starting from day 29, i.e. two weeks after the second immunization, hi one animal (#308) the SialylTn-specific IgG response was found only after the 5 ^th immunization. As expected, there was a 1-2 weeks delay in the onset of the anti-SialylTn IgG response compared to anti-SialylTn IgM response and IgG response against the carrier (Fig. 6A). In contrast, no SialylTn-specific IgG was induced in the group immunized without QS-21 adjuvant.

Fig. 6 Immune responses against carrier protein and SialylTn after immunization with SialylTn- mAbl7-lA vaccine with or without co-formulation with QS-21

Rhesus monkeys, (four animals per group) were immunized with SialylTn-mAbl7-lA vaccine with (A) or without (B) QS-21 adjuvant at days 1, 15, 29, 57 and re-boosted at day 226. Pre-sera and immune sera were analyzed for immune response by ELISA. The kinetics of the immune responses, i.e. antibody titers (geo mean and scatter factor) against SialylTn (IgG, IgM) and mAbl7-l A (IgG) are shown. Statistics: * p < 0.05 vs. Pre-serum (one-tailed, paired t-test).

Example 6:

Specificity of induced anti-SialylTn immune response

Methods:

ELISA for immune reactivity against mAbl7-lA

Pre-sera and immune sera of Rhesus monkeys were analyzed regarding the induced immune response against mAbl7-lA by ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated with lOμg/ml mAbl7-lA. ELISA plates were blocked with 10% FCS in PBS (Ih, 37°C), and samples pre-diluted in PBS with 2% FCS were incubated for 1.5 h at 37°C. A positive control serum with known reactivity against mAbl7-lA was tested in parallel and used for normalization between different ELISA plates. For detection plates were incubated with a sheep anti-human IgG-(γ-chain)-HRP conjugate (1:2000, Chemicon) for 30min at 37°C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) was stopped by adding 50μl H ₂ Sθ4 (30%). Absorbance was read at OD492/620. The titer was defined as reciprocal serum dilution yielding an absorbance of OD=LO. Curve fitting was done using GraphPad Prism program version 4.0.

SialylTn-PAA ELISA

Pre-serum and immune sera of Rhesus monkeys were analyzed regarding the immune response against the synthetic SialylTn carbohydrate antigen by SialylTn-PAA ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated lOμg/ml SialylTn-PAA (Lectinity, Finland). ELISA plates were blocked with PBS containing 10% FCS for Ih at 37°C. Serum samples were pre- diluted in PBS containing 2% FCS and 5% glucose, and incubated for 2 h at 37°C. A positive control serum with known reactivity against SialylTn was used for normalization between different ELISA plates. For detection plates were incubated with mouse anti-human IgM-FIRP conjugate (1:2000, SB, Southern Biotechnology), or sheep anti-human IgG-(γ-chain)-HRP conjugate (1 :2000, Chemicon), respectively, for 30min at 37°C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) was stopped by adding 50μl H ₂ SO ₄ (30%). Absorbance was read at OD492/620. Titers were defined as reciprocal of serum dilutions yielding an absorbance of OD=LO and OD=O.5 for IgM and IgG, respectively. Curve fitting was done using GraphPad Prism version 4.0.

Depletion experiment:

Immune serum (IS) was depleted by incubation with Sepharose beads coupled to mAbl7-lA

(mAb), SialylTn-mAbl7-lA, SialylTn-HSA, HSA or LeY-HSA, respectively,

Pre-serum (PS) or immune serum (IS), or immune serum after depletion were measured for antibody titers to SialylTn (A) or mAbl7-l A (B) by ELISA.

Results:

Specificity of anti-SialylTn response and anti-carrier responses

Specificity of anti-SialylTn response induced by SialylTn-mAbl7-lA immunizations of Rhesus monkeys was tested by depletion experiments: Immune serum (IS) was incubated with Sepharose beads coupled with either SialylTn-mAbl7-lA, mAbl7-lA, SialylTn-HSA, HSA or LeY-HSA, respectively. Pre-sera (PS) or immune sera (IS) or depleted immune sera were analyzed for binding to SialylTn in a SialylTn-PAA ELISA. The induced immune response measurable in the IS was significantly decreased by depletion with SialylTn-mAbl7-lA or SialylTn-HSA Sepharose beads, but not with mAbl7-l A, or HSA or HSA-LeY Sepharose beads where HSA was coupled to the unrelated LeY carbohydrate antigen and used as control (Fig. 7A).

Specificity of anti-carrier, i.e. anti-mAbl7-lA immune response induced by SialylTn-mAbl7- IA immunizations of Rhesus monkeys was also tested by depletion experiments: Immune serum (IS) was incubated with Sepharose beads coupled with either SialylTn-mAbl7-lA, mAbl7-lA, SialylTn-HSA, HSA or LeY-HSA, respectively. Untreated pre-sera (PS) or immune sera (IS) or depleted immune sera were analyzed for binding to mAbl7-lA by ELISA. The induced immune response measurable in the IS was found to be significantly decreased by depletion with mAbl7- IA or SialylTn-mAbl7-lA Sepharose beads, but not with HSA-SialylTn, or HSA or HSA-LeY Sepharose beads (Fig. 7B).

Fig. 7 Specificity of immune responses against SialylTn and mAbl7-lA carrier

The specificity of the immune responses against SialylTn carbohydrate antigen (A) and mAb 17- IA carrier protein (B) was demonstrated by depletion experiments. Pre-serum (PS) or immune serum (IS), or immune serum depleted by incubation with Sepharose beads coupled to mAb 17- IA (mAb), SialylTn-mAbl7-lA, SialylTn-HSA, HSA or LeY-HSA, respectively, were measured for antibody titers to SialylTn (A) or mAb 17- IA (B) by ELISA. Summarized data (geomean and scatter factor) of four Rhesus monkeys are shown. Statistics: * p < 0.05 vs. IS and ** p < 0.01 vs. IS (one-tailed, paired t-test)

Example 7:

Reactivity of induced immune response with natural targets: Ovine submaxillary mucin (OSM)

Methods:

Ovine submaxillary mucin (OSM) ELISA

Pre-sera and immune sera of Rhesus monkeys were analyzed regarding the immune response to OSM which is a natural substrate highly expressing SialylTn. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated with lOμg/ml OSM. ELISA plates were blocked with 10% FCS for Ih at 37°C, followed by a next washing step. Samples were pre-diluted in PBS with 2% FCS, and incubated for 2 h at 37°C. A positive control serum with known reactivity against OSM was tested in parallel and used for normalization between different ELISA plates. For detection plates were incubated with mouse anti-human IgM-HRP conjugate (1 :2000, SB, Southern Biotechnology), or sheep anti-human IgG-(γ-chain)-HRP conjugate (1:2000, Chemicon), respectively, for 30min at 37°C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) was stopped by adding 50μl H ₂ SO ₄ (30%). Absorbance was read at OD ₄₉₂ Ze ₂₀ . Titers were defined as reciprocal of serum dilutions yielding an absorbance of OD = 1.0 and OD = 0.5 for IgM and IgG, respectively. Curve fitting was done using GraphPad Prism version 4.0.

Results:

Significant antibody titers against OSM (IgM) were detectable in immune serum of all Rhesus monkeys immunized with SialylTn-mAbl7-lA plus QS-21 vaccine. Sera titers for IgM peaked after the second immunization. In three out of four animals also low IgG titers against OSM were induced after 3 ^rd immunization (data not shown).

Example 8:

Reactivity of induced immune response with natural targets: binding to SialylTn positive tumour cells (FACS analysis)

Methods:

Immune response against tumour cells (FACS analysis):

Binding of immune sera of Rhesus monkeys to tumour cells was measured by cell surface staining using a FACScan (Becton Dickinson). The OVCAR-3 ovary adenocarcinoma cells

(ATCC, HTB-161) were incubated with serum (diluted 1:40 in PBS with 2% FCS) for 2h on ice. For detection a goat F(ab')2 anti-human IgG (H+L)-PE conjugate (1:100, Immunotech, Marseille, France) was used. Pre- and immune sera were analyzed in parallel. Background binding of pre-sera was set at 10% positive cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

Results:

The immune response against the natural target, SialylTn positive tumour cells was tested. Pre- serum and immune sera of Rhesus monkeys immunized with SialylTn-mAbl7-lA plus QS-21 were analyzed for binding to SialylTn positive OVCAR-3 cells by FACS analysis. Histograms of IgG and IgM binding to OVCAR-3 cells are shown for SialylTn-mAbl7-lA vaccine (Fig. 8A). A statistically significant increase in cell binding to OVCAR-3 cells (both IgG and IgM) was found (p<0.05, paired t-test) (Fig. 8B).

Fig. 8 Induced reactivity against SialylTn positive tumour cells (FACS analysis)

Rhesus monkeys were immunized with SialylTn-mAbl7-lA vaccine plus QS-21. Pre-serum and immune sera were analyzed for binding to SialylTn positive OVCAR-3 tumour cells by FACS analysis. (A) Histograms for IgM (left panel) and IgG (right panel) binding to OVCAR-3 cells are shown, with immune serum (black line) superimposed on pre-serum (grey filled line). Dotted line: unstained control.

(B) Pre-serum and immune sera were analyzed for binding to SialylTn positive OVCAR-3 tumour cells by FACS analysis. Data of four Rhesus monkeys are shown. Statistics: *p<0.05 vs. Pre-serum (one-tailed, paired t-test).

Example 9:

Reactivity of induced immune response with natural targets: antibody-dependent cellular cytotoxicity (ADCC) against SialylTn positive tumour cells

Methods:

Antibody-dependent cellular cytotoxicity (ADCC) measured by Chromium release cell lysis assay

Pre-sera and immune sera of four Rhesus monkeys immunized with SialylTn-mAbl7-lA vaccine were tested for ADCC against SialylTn positive OVCAR-3 cells. Human PBMCs were used as effector cells and incubated with the target cells at two E:T ratios, i.e. 60:1 and 20:1 for 14h. ⁵¹ Cr -release was measured by a γ-counter.

Results:

While different initial levels of lytic activity were found in the different animals, a clear increase in ADCC activity was found in the immune sera (IS) of all animals in comparison to the corresponding pre-sera (PS) (Fig. 9).

Fig. 9 ADCC of induced immune response in SialylTn-mAbl7-lA multi-component vaccine against SialylTn expressing OVCAR-3 tumour cells

Rhesus monkeys were immunized with the SialylTn-mAbl7-lA vaccines. Pre-serum and immune serum was tested for ADCC against SialylTn positive OVCAR-3 cells. Human PBMCs were used as effector cells and incubated with the ⁵¹ Cr-labeled target cells at E:T ratios, i.e. 60:1 and 20:1 for 14h. ⁵¹ Cr -release was measured by a γ-counter.

Example 10:

Reactivity of induced immune response with natural targets: binding also to SialylTn negative tumour cells (FACS analysis) - correlation with reactivity against the carrier

Methods:

Immune response against tumour cells (FACS analysis):

Binding of immune sera of Rhesus monkeys to tumour cells was measured by cell surface staining using a FACSCalibur (Becton Dickinson). The KATOIII tumour cells were incubated with serum (diluted 1:40 in PBS with 2% FCS) for 2h on ice. For detection a goat F(ab')2 anti- human IgG (H+L)-PE conjugate (1:100, Immunotech, Marseille, France) was used. Pre- and immune sera were analyzed in parallel. Background binding of pre-sera was set at 10% positive

cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

Results:

An immune response also against tumour cells, KATOIII which are negative for SialylTn, was detected as analysed by FACS analysis. A statistically significant increase in cell binding to KATOIII tumour cells (IgG) was found (p<0.05, paired t-test).

The kinetics of the detected KATOIII binding reactivity induced by immunization correlates with the kinetics of the induced immune response against the carrier molecule (see Fig. 6)

Fig. 10

Cell binding of induced immune response to KATOIII tumour cells.

Rhesus monkeys were immunized with IGN402. Pre-serum and immune sera were analyzed for binding to KATOIII tumour cells by FACS analysis. Data of four Rhesus monkeys are shown.

Statistics: *p<0.05 vs. Pre-serum (one-tailed, paired t-test).

Example 11 :

Cytokine release in serum after vaccination

Methods: Cytokine release (xMAP Luminex Multiplex):

Pre-sera and immune sera were analyzed for cytokines (IL- lβ, IL-2, IL-4, IL-6, IL-8, IL-IO, IL- 12(p70), TNFα, IFNγ and GM-CSF) by xMAP Multiplex technology (Luminex) using the Beadlyte Human Multi-Cytokine Detection System 3 (Upstate) according to the manufacturer's protocol. Briefly, 25 μl of serum sample were incubated with 25μl assay buffer and a mixture of anti-human cytokine antibodies coupled to beads for 2 hours at 25°C in the dark. After addition of biotinylated anti-human cytokine detection antibodies and incubation for 1.5 hours, the liquid was removed and streptavidin-phycoerytrin was added. Subsequently, the beads were washed with assay buffer. The emitted fluorescent signal (MFI) was measured using a Luminex 100 reader (settings: 50 events per bead, 50μl sample size, gate : 7500-13000 MFI).

Results:

Cytokine release in serum after repeated vaccination

The serum cytokine profile during the time course of the immunizations was measured using xMAP Multiplex technology (Fig 11). Apart from detectable levels of the chemokine IL-8, no significant cytokine levels were found in the pre-serum (i.e. before immunization) of healthy Rhesus monkeys. Also following the first immunization no significant cytokine levels were measured. Starting from day 22, i.e. one week following the second immunization, low amounts of IFNγ could be detected. Moreover, following the 3 ^rd and 4 ^th immunizations (day 43 and day 71, respectively) significant levels of IFNγ, IL-8, IL- lβ, TNFoc, IL-2, GM-CSF and IL-4, were measured in the serum of animals immunized with the vaccine plus QS-21 adjuvant (Fig. HA and C). Low levels of IL-6 and IL- 12 were also found in some of the animals, but in none of the animals IL-IO was measured during vaccination. In contrast, immunization with the vaccine without QS-21 resulted in marginal cytokine release only, with some IFNγ and IL-8 measurable after the 3 ^rd and 4 ^th immunizations. Interestingly, following a re-boost (5 ^th ) immunization, half a year later, in two out of four animals immunized with the vaccine without QS-21 significant cytokine levels were found, although cytokine levels were generally lower than in the QS-21 group (Fig. 11 B,D vs. A,C).

Correlation of time kinetics of SialylTn-specific IgG response and cytokine release

Comparing the kinetics for induction of immune responses against carrier molecule (IgG) or the SialylTn carbohydrate antigen (IgM, IgG) (Fig 6) with the kinetics for cytokine release (Fig 11) suggested a timely correlation between IgG switch induction against SialylTn and the cytokine release measurable in serum. For more detailed analysis, for each animal the kinetics of the immune responses against the carrier and the SialylTn carbohydrate antigen, respectively, and the kinetics of the cytokine release were superimposed on the same time scale (Fig. 12). The absolute values for IgG titers (against carrier and SialylTn) were normalized by a multiplication factor as indicated to fit the scale. Neither induced SialylTn-specific IgG antibodies nor measurable cytokines were found during the first two immunizations. In contrast, after the 3 ^rd and 4 ^th or 5 ^th immunizations along side with high titer IgG responses against the carrier molecule also detectable IgG responses against the SialylTn carbohydrate antigen were found. Noteworthy, the induction of the anti-SialylTn IgG response coincided with the temporary cytokine release measurable in the immune serum. A particularly consistent correlation in timing was evident for the SialylTn-specific IgG switch induction and the release of IL-2, IL- l β and

IFNγ (Fig. 12A-D). In one animal of this group, #308, the IgG switch against SialylTn was induced only after the 5th re-boost immunization, again correlating with a pronounced cytokine release (Fig. 12C).

Fig. 11 : Cytokine release after immunization with SialylTn-mAbl7-l A vaccine with or without QS-21 adjuvant

Rhesus monkeys (four animals per group) were immunized with SialylTn-mAbl7-lA vaccine with (A, C) or without (B, D) QS-21 adjuvant at days 1, 15, 29, 57 and re-boosted at day 226. Pre-serum and immune sera were analyzed for cytokine release by xMAP technology (Luminex). Cytokine levels (pg/ml) measured in serum are shown (mean and SD).

Fig. 12: Kinetics of immune response against SialylTn coincides with a temporary cytokine release in serum

Rhesus monkeys were immunized with SialylTn-mAbl7-lA vaccine co-formulated with lOOμg QS-21 adjuvant at days 1, 15, 29, 57, and re-boosted at 24 weeks after last immunization at d226. Pre-serum (d-7, d 1) and immune sera (dl5, 22, 29, 43, 57, 71, 85, 99, 226, and 240) were analyzed for reactivity against SialylTn (IgG) or mAbl7-lA carrier (IgG) by ELISA and cytokine release in the serum was measured by xMAP Multiplex technology (Luminex). Antibody titers and cytokine levels (pg/ml serum) of each individual Rhesus monkey (A, B, C, D, respectively) are shown. The indicated multiplication factor was used to align antibody titer values with the cytokine levels (pg/ml) - figure scales are adjusted to cytokine levels.

Example 12

Activation of antigen un-specific effector cells, such as NK cells results in tumour cell lysis

(irrespectively on antigen expression)

Methods:

In vitro activation of human NK cells by immune serum

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors (obtained from the Austrian Red Cross) on a Ficoll density gradient. The NK cell enriched fraction was obtained from the non-adherent PBMC fraction (after removal of adherent

cells by 1 h plastic adherence) and by negative sorting on an AutoMACS ™ using antibodies against CD3, CD14, CD34, and CD19. The NK cell enriched population was characterized by FACS analysis using CD56 / CD 16 as marker and consisted of at least 60 % NK cells. The NK-enriched cell fraction was incubated with pre-serum or immune serum (at a final dilution of 1 :5), and in the presence or absence of supplements of indicated amounts of hIL-2, for 48 h. The medium was replaced with fresh RPMI 1640 containing 10 % FCS and incubation was continued for another 24 h. After washing the cells were re-suspended in fresh medium and used as effector cells in a 4 h ⁵¹ Cr-release lysis assay against labeled KATOIII tumour target cells at effector to target (E:T) ratios of 10:1 and 1:1, respectively. Release of ⁵¹ Cr from lysed target cells into the supernatant of the samples ("Cs") was measured using a γ-counter (Cobra 5005, Canberra-Packard, Australia). Spontaneous release ("Sr") and maximum release (100 %, "Mr") were measured after incubation of target cells with medium alone or with detergent (2 % SDS), respectively. Cytotoxicity was calculated using the formula 100 % x (Cs-Sr) / (Mr-Sr).

Ex vivo measurement of activation of non-adherent PBMCs derived from Rhesus monkeys before and after immunization

PBMCs were isolated from heparinized blood derived from Rhesus monkeys before or after immunization, respectively, on a Ficoll density gradient. After washing, the non-adherent PBMC fraction was obtained by removal of adherent cells following 2 h plastic adherence. Nonadherent PBMC were incubated in RPMI 1640 supplemented with 10 % FCS for 2 h and subsequently used as effector cells in an overnight ⁵¹ Cr-release lysis assay with labeled KATOIII target cells at the E:T ratios of 60:1, 30:1, 10:1 and 3:1. Cytotoxicity was calculated as described above.

Results:

In vitro activation of human NK activity by pre-incubation with immune serum

To elucidate whether the induced cytokines may have triggered further biological effects, the potential activation of NK cells was investigated. Natural killer (NK) cells are known to kill target cells in a MHC independent manner and without re-exposure to antigen. NK cells have been shown to be activated by cytokines such as IL- 1 , IFNγ and IL-2 and can be induced by high dose IL-2 to obtain feature of Lymphokine-Activated Killer (LAK) cells. Our preliminary experiments using enriched human NK cells showed efficient lysis of the NK sensitive target

cells K562, but only marginal efficacy against the NK resistant DAUDI cells or KATOIII tumour cells. In contrast, pre-incubation of the NK cells with high doses of recombinant IL-2 (1000 U/ml) resulted in pronounced lysis of DAUDI cells and KATOIII cells indicating the generation of LAK cells (data not shown). To test the effect of released cytokines on NK effector functions, the NK enriched cell fraction of human PBMCs was incubated for two days with 1 :5 diluted pre-serum or immune serum derived from Rhesus monkeys following repeated boost immunizations (at time points of the highest measured cytokine levels). In order to compensate for potential inhibitory factors present in the Rhesus monkey serum, pre-serum and immune serum were used either alone or supplemented with a moderate IL-2 dose (100 U/ml) which by itself was not sufficient to induce sufficient activation of NK cells to lyse DAUDI or KATOIII tumour cells (data not shown). Incubation of effector cells with immune serum enhanced the lytic activity of NK enriched human PBMCs against KATOIII target cells in three out of four animals (Fig. 13).

Rhesus monkey derived PBMCs show enhanced cytolytic activity against tumour cells after immunization

In order to test whether the in vitro activation of NK enriched PBMC by pre-incubation with immune serum (Fig. 12) is reflecting also a stimulation of cellular cytotoxicity in vivo, PBMCs derived from Rhesus monkey before and after immunization, respectively, were tested for their lytic effect on ⁵¹ Cr labeled KATOIII tumour cells at different E:T ratios. Before being used as effector cells the PBMCs were depleted of monocytes by plastic adherence and any Rhesus monkey derived serum was removed by repeated washing steps. The data show that in all four tested Rhesus monkeys the cytolytic activity of their PBMCs was enhanced following repeated immunization of the animals compared to the time points before immunization, i.e. day 1 (Fig. 14).

Fig. 13: NK cell mediated lysis of KATOIII tumour cells after in vitro activation of human NK cells by immune serum of immunized Rhesus monkeys

Rhesus monkeys were immunized with SialylTn-mAbl7-lA vaccine co-formulated with QS-21. NK cells derived from PBMCs (obtained after negative sorting by CD3/CD14/CD34/CD19) were incubated with pre-serum (PS) or immune serum (IS, with peak cytokine levels) for 48h. After removal of medium, washed cells were incubated for additional 24 h. Activated NK cells

were used as effector cells in a 4 h ⁵¹ Cr-release lysis assay against labeled KATOIII target cells. Data of three individual animals, each measured in independent triplicates (mean and SD shown), are presented (Panels A, B, C). Statistics: * p < 0.05 vs. respective Pre-serum control (one-tailed, paired t-test).

Fig. 14: Lysis of KATOIII tumour cells by Rhesus monkey derived PBMCs taken after repeated boost immunizations

Rhesus monkeys were immunized with SialylTn-mAbl7-lA vaccine co-formulated with QS-21. PBMCs were isolated from heparinized blood derived from Rhesus monkeys before or after immunization, respectively, on a Ficoll density gradient. Following removal of adherent cells by 2 h plastic adherence and repeated washing steps, the non-adherent PBMCs were used as effector cells in an overnight ⁵¹ Cr-release lysis assay against labeled KATOIII target cells. Data of four individual animals (#31, #48, #76, #318), each measured in independent triplicates (mean and SD shown), are presented. Statistics: * p < 0.05 vs. Pre-serum (dl), (one-tailed, paired t-test).

Example 13

Coupling of a Lewis Y Carbohydrate to an EpCAM-Specific Antibody, mAbl7-lA (also designated as HE2)

Methods:

The antibody HE2 is described in the patent application WO 00/41722 and upon an appropriate immunization is able to induce an immune response binding to tumour cells. According to the invention, a synthesized Lewis Y carbohydrate antigen is coupled to mAbl7-lA. In this example, coupling was performed by a chemical method:

The antibody mAbl7-lA is coupled to N-hydroxysuccinimide-activated synthetic Lewis Y tetrasaccharide (Syntesome GmbH, Munich, Germany) in a suitable buffer (100 mM sodium phosphate buffer containing 150 mM NaCl, pH 8.5). N-hydroxysuccinimide-activated Lewis Y- tetrasaccharide is dissolved in N,N-dimethylformamide (100 mg/ml) and added drop- wise to an mAbl7-lA antibody solution in the appropriate buffer (100 mM sodium phosphate puffer containing 150 mM NaCl, pH 8.5) and shaken for at least 2.5 hours at 4C. The extent of glycosylation of the antibody with Lewis Y can be controlled by selecting the molar excess of

activated sugar as well as the concentration of antibody-containing solution (1-10 mg/ml). For comparative purposes, two different reaction batches are prepared by varying the molar excess

(5-fold and 15-fold, respectively) of activated sugar: "neoglycoprotein I" having a lower carbohydrate portion and "neoglycoprotein II" having a higher carbohydrate portion.

The bispecificity of the neoglycoprotein can be detected by various immunological methods

(ELISA or Western blotting with antibodies directed against the Lewis Y determinant or against mAbl7-lA).

Direct ELISA: mAbl7-lA, mAb 17-1 A/Lewis- Y neoglycoprotein or LeY-PM (polyacrylamide-coupled tetrasaccharide, Syntesome 045-PA) is dissolved in a coating buffer (15 mM Na2CO3, 5 mM NaHCCh, 3 mM NaN3, pH 9.6) at 10 μg/ml an adsorbed onto a microtiter plate (Nunc Maxisorb, Denmark) (1 hour at 37C, 100 μl/well). After washing three times of the microtiter plate wells with washing buffer (2% NaCl, 0.2% Triton X-100 in PBS) unspecific binding sites are blocked with 200μl blocking buffer (5% fetal bovine serum in PBS (138 mM NaCl, 1.5 mM KOH, 2.7 mM KCl, 6.5 mM Na ₂ HPO ₄ , pH 7.2) for 30 minutes at 37C and subsequently, after washing trice, incubation with specific anti-Lewis Y antibody (human) or goat anti- mAb 17- IA antibody (1 μg/ml) dissolved in dilution buffer (2% FCS in PBS) 100 ml was effected for 30 minutes at 37C. Unbound antibodies are removed by three-time washing with washing buffer. The bound antibodies are detected by an HRP conjugate specific for the respective detection antibody (goat anti-human IgG+ A+M HRP of Zymed (USA) for anti-Lewis Y antibody; mouse anti-goat IgG HRP (Axell, USA) for anti-HE2 antibody (1 μg/ml) for 30 minutes at 37C. After subsequent washing (3 times with washing buffer and once with staining buffer), the staining of 100 μl orthophenylene diamine dihydrochloride solution (Sigma, USA; dissolved in staining buffer and activated with H2O2; 30%, Merck, Germany) is initiated by bound HRP conjugate and the color development is stopped with 15% sulfuric acid (50 μl). On a microplate photometer (Labsystem, Model No. 354), the developing color intensity is measured at 492 nm with the reference wavelength being 620 nm. After a further washing step with staining buffer (24.3 mM citric acid, 51.4 mM Na ₂ HPO ₄ , pH5).

Sandwich ELISA:

Human anti-Lewis Y antibody (10 mg/ml) dissolved in coating buffer; 1001) is nonspecifically bound to a microtiter plate (Nunc, Maxisorb) (1 hour incubation at 37C), after washing trice with

washing buffer is blocked with 200μl 5% fetal bovine serum in PBS (incubation for 30 minutes at 37C) and incubated with mAbl7-lA -Lewis Y-neoglycoproteins I and II as well as mAbl7- IA as a control in various concentrations (1.25-7.63*10 ^"6 g/ml; 100 μl) for 1 hour at 37C. After three-time washing in washing buffer, incubation is effected with goat anti- mAbl7-lA antibody (1 mg/ml in dilution buffer; 100 [mu]l) for 30 minutes at 37C. Excess antibodies are removed in a subsequent washing step (3 times with washing buffer). Bound antibodies are recognized by incubation (30 minutes, 37C) with mouse anti-goat IgG HRP (Axell, dissolved 1:1000 in dilution buffer, 100 μl): After subsequent washing (3 times with washing buffer, once with staining buffer), bound HRP conjugate triggers a staining reaction of 1001 added orthophenylene diamine dihydrochloride solution (Sigma, 10 mg dissolved in 25 ml staining buffer and activated with 10 μl H2O2; 30%, Merck). The color reaction is stopped with 50 μl 15% H ₂ SO ₄ and the extinction is measured at 492 nm (reference wavelength 620 nm) on a microplate photometer (Labsystem, Model No. 354).

Results:

The coupling products are detected distinguishable from the starting material

Fig. 15: Analysis of coupling products by Western Blot:

As with SDS-PAGE, the samples are separated according to size. After this, the separated proteins are blotted on a nitrocellulose membrane, blocked for an hour in 3% milk powder solution and subsequently incubated with human anti-Lewis Y antibody (10 μg/ml in PBS) for two hours. Bound antibodies are detected by goat anti-human HRP conjugate (1:500 in PBS) specific for anti-Lewis Y antibody. The Lewis Y glycosylated proteins are visualized by a subsequent color reaction.

As is apparent from FIG. 14, coupling products with anti-Lewis Y antibody (left side), HE2 is used synonym to mAbl7-lA in this Figure 14

Example 14

Use of Immunogenic anti-idiotypic Antibody (imitating Lewis Y) as carrier

Various anti-idiotypic antibodies have been tested in clinical trails demonstrating the feasibility of this approach. For example, the anti-idiotypic murine antibody MMA383, i.e. IGN301, which contains within its antigen binding site a Lewis-Y mimic, is a potential candidate for the carrier

molecule. Active application of this antibody induces an immune response that contains antibodies capable of binding to Lewis Y moieties attached to extra-cellular (and cellular) proteins and peptides (Fig.16). IGN301 has been tested in a Phase Ib trail in the US and showed a good safety profile.

In general, using any anti-idiotypic antibody as carrier has the advantage that this immunogenic molecule provides already an epitope against which therapeutic antibodies are induced. Furthermore, such carrier can subsequently be conjugated to e.g. SialylTn and pre-selected, immunogenic peptides (e.g. derived from EpCAM, or Her2-neu, or EGF-receptor) generating a true multi-epitope vaccine. Immunogenic peptides derived from known TAAs antigens may be identified by epitope mapping experiments or by rational design (e.g. by the antigenic index). After active application of the multi-epitope vaccine, the induced polyclonal, tri-specific immune response should have a significantly increased success rate in targeting (and killing) tumour cells (Fig. 16).

Fig. 16

Anti-idiotypic antibodies, mimicing tumour associated antigens (carbohydrates or peptides), as immunogenic carrier. Graphical depiction of the immunological concept. Symbols: Blue = Lewis-Y mimic, Red = coupled Sialyl-Tn, Green = coupled EpCAM derived epitope

Detailed description of example:

Coupling of SialylTn Carbohydrate to an Anti-idiotypic Antibody (MM A383), and Formulation of the multi-component vaccine

Methods:

Coupling of SialylTn carbohydrate to MMA383

The SialylTn carbohydrate antigen is coupled to the MMA383 (murine IgGl) protein carrier at a molar ratio of SialylTn to MM A383 of 18:1 by reacting 10 mg of nitrophenylated spacered

SialylTn, Neu5Acα2-6GalNAcα-O(CH ₂ )3NHCO(CH2)4COO-(p-NO ₂ C6H4) (MW 819 g/mol,

Lectinity, Finland), with lOOmg of MMA383. Briefly, 100 mg of MMA383 (lOmg/ml) is dialyzed twice at 4°C for 20 h against 700 ml Coupling Buffer (0.1 M Na-PO ₄ , 0.15 M NaCl, pH

8.5) using a Slide- A-Lyzer dialysis cassette MWCO 1OK (Pierce). Concentration of MMA383 is determined by Size Exclusion Chromatography (SEC). In parallel, 10 mg of Neu5Acα2-

6GalNAcψ-O(CH ₂ )3NHCO(CH2)4COO-(p-Nθ2C ₆ H4) are dissolved in 300 μl DMF and added to the ice cold MMA383. The reaction mixture is incubated rotating at +4°C. The kinetic of the reaction is monitored by the size of the coupling product as analyzed by SEC. After 28 h the reaction mixture is dialyzed against Formulation Buffer (1 mM Na-P(X, 0.86% NaCl, pH 6.0) using Slide-A-Lyzer dialysis Cassette 3.5K (Pierce) at + 4°C for 2Oh. For comparison, uncoupled MMA383 is processed in parallel. SialylTn-MMA383 coupling products are analyzed by size exclusion chromatography, LDS-PAGE, Western blot, isoelectric focusing (IEF), and Resorcinol assay.

Analysis of coupling products (tumour-antigen-carrier conjugates)

Size Exclusion Chromatography

Concentrations of SialylTn-MMA383 coupling products are quantified by size exclusion chromatography (SEC) on a ZORBAX GF-250 column in a Dionex system and on a TSKgel G3000SW column in a HPl 100 system.

IEF, LDS-PAGE and Western Blots

SialylTn-MMA383 coupling products are analyzed by SERVALYT PRECOTES horizontal flat bed IEF electrophoresis pH 3-10 (SERVA) followed by Coomassie blue staining (Invitrogen), and lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE, NuPAGE Electrophoresis System, Bis-Tris-Gel, 4-12%) under reducing conditions (DTT, 1,4- Dithiothretiol) followed by SilverXpress™-staining (Invitrogen).

For Western blot analysis, samples following LDS-PAGE are transferred (25V, 1.1 W, 1.5h) to Immobilon membranes (PVDF 0.45 μm, Millipore). Membranes are blocked with 3% skim milk and stained with rabbit anti-mouse IgG (H+L)-HRP (1:1000, Zymed) or alternatively with anti- SialylTn CD175s (IgGl) (lOμg/ml, DAKO) conjugated to HRP.

Quantification of Sialic acid by Resorcinol assay

Amount of sialic acid in the coupling products is quantified by Resorcinol-HCl reaction in the presence of CuSO4, and spectrophotometric measurement of absorbance at OD562. Briefly, 0.15 ml of samples and 0.15ml Resorcinol reagent (2% Resorcinol, 2.5mM CUSO4, HCL (37%)) are mixed in tightly closed glass vials, and incubated at 100°C for Ih. The color reaction is measured by adsorption at OD5 ₆ 2 (Spectrophotometer Ultrospec II, LKB Biochrom). As a standard a

'SialylTn mix' (consisting of Sialic acid (i.e. N-Acetylneuraminic acid) and GaINAc at a molar ratio 1:1), 1.25 - 25 μg/ml is used for calibration.

Multi-component vaccine Formulation

500 μg of SialylTn-MMA383 conjugate are adsorbed on 1.67 mg aluminum hydroxide in 0.5 ml formulation buffer (ImM Na-PO4, 0.86% NaCl, pH 6.0). The vaccine is formulated either w/o additional adjuvant or co-formulated with lOOμg QS-21 adjuvant (Antigenics Inc., Lexington,

MA).

LAL Assay

Amounts of endotoxin in the vaccine formulations are estimated by Limulus Amebocyte Lysate - Endochrome™ assay (Charles River Endosafe) according to the manufacturer. None of the final vaccine formulations should contain significant amounts of endotoxin.

Pyrogenicity test in rabbits

Final vaccine formulations are subjected to pyrogenicity test by i.v. application in rabbits. The formulations used for this study should be negative regarding pyrogenicity testing.

Results:

Nitrophenyl-activated, spacered SialylTn is reacted with MMA383 at a molar ratio of 18:1. The kinetic of the reaction is monitored by SEC, and the reaction is stopped when no further increase in the size of the coupling product is detected. The SialylTn-MMA383 coupling product is analyzed by LDS-PAGE under reducing conditions followed by Silver staining (and Western blotting stained with anti-SialylTn CDl 75s (IgGl) - HRP. LDS-PAGE is expected to show an increase in the molecular weight of the heavy chains (5OkDa) and the light chains (25kDa) in the coupling products compared to uncoupled MMA383, indicating that SialylTn has been coupled to both, heavy and light chains of the MMA383. Western blot analysis is expected to show that SialylTn is detectable in the SialylTn-MMA383 coupling products but not in the MMA383. Coupling of the (negatively charged) SialylTn to MMA383 is expected to result in a shift of the isoelectric point as shown by IEF. The molar ratio of SialylTn to MMA383 in the coupling product is quantified using the Resorcinol assay. In this assay the amount of sialic acid in the coupling products is quantified by Resorcinol-HCl reaction in the presence of CuS(X

The SialylTn-MMA383 coupling product (500μg) is adsorbed onto aluminum hydroxide, aliquoted into sterile vials. The aluminium adsorbed multi-component vaccine is used either w/o additional adjuvant or in a co-formulation with lOOμg QS-21 adjuvant. The vaccine formulations are analyzed for traces of endotoxin by Limulus Amebocyte Lysate and pyrogenicity test by i.v. application in rabbits.

Immunization of Rhesus monkeys with SialylTn-MMA383 multi-component vaccine and analysis of the induced immune responses against carrier and coupled tumour-antigen (SialylTn): Elucidation of the effect of additional adjuvant (QS-21)

Methods:

Rhesus monkey immunization study

Safety, tolerability and immunogenicity of multiple subcutaneous injections of IGN402 are evaluated in vaccination studies in Rhesus monkeys. All animal studies are performed under controlled and documented conditions in accordance with animal health care standards. Per group, four healthy adult Rhesus monkeys (age and sex matched, are vaccinated on days (d) 1,

15, 29 and 57 by subcutaneous injection and re-boosted on a later time point. Blood samples are taken before and after immunization for serum analytic.

In a second Rhesus monkey study animals - four animals per group are vaccinated with four initial immunizations. Heparinized blood samples are taken before and after immunization for preparation of PBMC.

ELISA for immune reactivity against MMA383

Pre-sera and immune sera of Rhesus monkeys, i.e. sera obtained before and after immunization, respectively, are analyzed regarding the induced immune response against MMA383 by ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) are coated with 10 μg/ml MMA383. Wells are blocked with 10 % FCS in PBS for Ih at 37°C and samples pre-diluted in PBS with 2 % FCS are incubated for 1.5 h at 37°C. A positive control serum (derived from a Rhesus monkey immunized with MMA383 formulated on alhydrogel) with known reactivity against MMA383 is tested in parallel and used for normalization of OD values between different ELISA plates. For detection plates are incubated with a 1:2000 diluted sheep anti-human IgG-(γ-chain)-HRP conjugate (Chemicon) for 30 min at 37°C. Staining with substrate OPD (10 mg OPD dissolved in 25 ml staining buffer containing 10 μl 30 % H ₂ O ₂ ) is stopped by adding 50 μl H ₂ SO ₄ (30 %)

and the color intensity is measured at OD492/620. The titer is defined as reciprocal serum dilution yielding an absorbance of OD = 1.0 on a titration curve. Curve fitting is done using GraphPad Prism version 4.0.

SialylTn-PAA ELISA

Pre-serum and immune sera of Rhesus monkeys are tested for reactivity against the synthetic SialylTn carbohydrate antigen by SialylTn-PAA ELISA. Briefly, ELISA plates (¥96 Maxisorp, NUNC) are coated lOμg/ml SialylTn-PAA (Lectinity, Finland). Wells are blocked with PBS containing 10 % FCS for 1 h at 37 ⁰ C. Serum samples are pre-diluted in PBS containing 2 % FCS and 5 % glucose, and incubated for 2 h at 37°C. A positive control serum with known reactivity against SialylTn is used for normalization between different ELISA plates. For detection plates are incubated with 1:2000 diluted mouse anti-human IgM-HRP conjugate (Southern Biotechnology) or 1:2000 diluted sheep anti-human IgG-(γ-chain)-HRP conjugate (Chemicon), respectively, for 30 min at 37°C. Staining with substrate OPD (10mg OPD dissolved in 25 ml staining buffer containing 10 μl 30 % H2O2) is stopped by adding 50 μl H2SO4 (30 %) and the color intensity is measured at OD492/620. Titers are defined as the reciprocal value of serum dilutions yielding an absorbance of OD = 1.0 and OD = 0.5 for IgM and IgG, respectively. Curve fitting is done using GraphPad Prism version 4.0.

Specificity of induced anti-SialylTn immune response

Methods:

ELISA for immune reactivity against MMA383

Pre-sera and immune sera of Rhesus monkeys are analyzed regarding the induced immune response against MMA383 by ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) are coated with lOμg/ml MMA383. ELISA plates are blocked with 10% FCS in PBS (Ih, 37°C), and samples pre-diluted in PBS with 2% FCS are incubated for 1.5 h at 37°C. A positive control serum with known reactivity against MMA383 is tested in parallel and used for normalization between different ELISA plates. For detection plates are incubated with a sheep anti-human IgG- (γ-chain)-HRP conjugate (1 :2000, Chemicon) for 30min at 37 ⁰ C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) is stopped by adding 50μl H ₂ SO ₄ (30%). Absorbance is read at OD ₄₉₂ /62o. The titer is defined as reciprocal serum dilution yielding an absorbance of OD = 1.0. Curve fitting is done using GraphPad Prism program version 4.0.

SialylTn-PAA ELISA

Pre-serum and immune sera of Rhesus monkeys are analyzed regarding the immune response against the synthetic SialylTn carbohydrate antigen by SialylTn-PAA ELISA. Briefly, ELISA plates (F96 Maxisorp, NUNC) are coated lOμg/ml SialylTn-PAA (Lectinity, Finland). ELISA plates are blocked with PBS containing 10% FCS for Ih at 37 ⁰ C. Serum samples are pre-diluted in PBS containing 2% FCS and 5% glucose, and incubated for 2h at 37 ⁰ C. A positive control serum with known reactivity against SialylTn is used for normalization between different ELISA plates. For detection plates are incubated with mouse anti-human IgM-HRP conjugate (1:2000, Southern Biotechnology), or sheep anti-human IgG-(γ-chain)-HRP conjugate (1:2000, Chemicon), respectively, for 30min at 37°C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) is stopped by adding 50μl H ₂ SO ₄ (30%). Absorbance is read at OD492/620. Titers are defined as reciprocal of serum dilutions yielding an absorbance of OD=LO and OD=O.5 for IgM and IgG, respectively. Curve fitting is done using GraphPad Prism version 4.0.

Depletion experiment:

Immune serum (IS) is depleted by incubation with Sepharose beads coupled to MMA383 (mAb), SialylTn-MMA383, SialylTn-HSA, HSA or LeY-HSA, respectively. Pre-serum (PS) or immune serum (IS), or immune serum after depletion are measured for antibody titers to SialylTn (A) or MMA383 (B) by ELISA.

Reactivity of induced immune response with natural targets: Ovine submaxillary mucin (OSM)

Methods:

Ovine submaxillary mucin (OSM) ELISA

Pre-sera and immune sera of Rhesus monkeys are analyzed regarding the immune response to

OSM which is a natural substrate highly expressing SialylTn. Briefly, ELISA plates (F96

Maxisorp, NUNC) are coated with lOμg/ml OSM. ELISA plates are blocked with 10% FCS for

Ih at 37°C, followed by a next washing step. Samples are pre-diluted in PBS with 2% FCS, and incubated for 2 h at 37°C. A positive control serum with known reactivity against OSM is tested in parallel and used for normalization between different ELISA plates. For detection plates are incubated with mouse anti-human IgM-HRP conjugate (1:2000, SB, Southern Biotechnology), or

sheep anti-human IgG-(γ-chain)-HRP conjugate (1:2000, Chemicon), respectively, for 30min at 37°C. Staining with substrate OPD (lOmg OPD dissolved in 25ml + lOμl 30% H ₂ O ₂ ) is stopped by adding 50μl H ₂ Sθ4 (30%). Absorbance is read at OD492/620. Titers are defined as reciprocal of serum dilutions yielding an absorbance of OD = 1.0 and OD = 0.5 for IgM and IgG, respectively. Curve fitting is done using GraphPad Prism version 4.0.

Reactivity of induced immune response with natural targets: binding to SialylTn positive tumour cells (FACS analysis)

Methods:

Immune response against tumour cells (FACS analysis):

Binding of immune sera of Rhesus monkeys to tumour cells is measured by cell surface staining using a FACScan (Becton Dickinson). The SialylTn expressing OVCAR-3 ovary adenocarcinoma cells (ATCC, HTB-161) are incubated with serum (diluted 1 :40 in PBS with 2%

FCS) for 2h on ice. For detection a goat F(ab') ₂ anti-human IgG (H+L)-PE conjugate (1:100,

Immunotech, Marseille, France) is used. Pre- and immune sera are analyzed in parallel.

Background binding of pre-sera is set at 10% positive cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

Reactivity of induced immune response with natural targets: binding to Lewis Y positive tumour cells (FACS analysis)

Methods:

Immune response against tumour cells (FACS analysis):

Binding of immune sera of Rhesus monkeys to tumour cells is measured by cell surface staining using a FACScan (Becton Dickinson). The LewisY expressing SKBR3 tumour cells (ATCC,

#HTB30) are incubated with serum (diluted 1:40 in PBS with 2% FCS) for 2h on ice. For detection a goat F(ab') ₂ anti-human IgG (H+L)-PE conjugate (1:100, Immunotech, Marseille,

France) is used. Pre- and immune sera are analyzed in parallel. Background binding of pre-sera is set at 10% positive cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

Reactivity of induced immune response with natural targets: antibody-dependent cellular cytotoxicity (ADCC) against SialylTn positive tumour cells

Methods:

Antibody-dependent cellular cytotoxicity (ADCC) measured by Chromium release cell lysis assay

Pre-sera and immune sera of four Rhesus monkeys immunized with SialylTn-MMA383 vaccine are tested for ADCC against SialylTn positive OVCAR-3 cells. Human PBMCs are used as effector cells and incubated with the target cells at two E:T ratios, i.e. 60:1 and 20:1 for 14h. ⁵¹ Cr -release is measured by a γ-counter.

Reactivity of induced immune response with natural targets: binding also to Lewis Y positive (SialylTn negative) tumour cells (FACS analysis) - correlation with reactivity against the carrier

Methods:

Immune response against tumour cells (FACS analysis):

Binding of immune sera of Rhesus monkeys to tumour cells is measured by cell surface staining using a FACScan (Becton Dickinson). The LewisY expressing KATOIII tumour cells or the

LewisY expressing SKBR-3 are incubated with serum (diluted 1:40 in PBS with 2% FCS) for 2h on ice. For detection a goat F(ab')2 anti-human IgG (H+L)-PE conjugate (1:100, Immunotech,

Marseille, France) is used. Pre- and immune sera are analyzed in parallel. Background binding of pre-sera is set at 10% positive cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

Cytokine release in serum after vaccination

Methods: Cytokine release (xMAP Luminex Multiplex):

Pre-sera and immune sera are analyzed for cytokines (IL- lβ, IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12(p70), TNFα, IFNγ and GM-CSF) by xMAP Multiplex technology (Luminex) using the Beadlyte Human Multi-Cytokine Detection System 3 (Upstate) according to the manufacturer's protocol. Briefly, 25 μl of serum sample are incubated with 25 μl assay buffer and a mixture of anti-human cytokine antibodies coupled to beads for 2 hours at 25°C in the dark. After addition

of biotinylated anti-human cytokine detection antibodies and incubation for 1.5 hours, the liquid is removed and streptavidin-phycoerytrin is added. Subsequently, the beads are ished with assay buffer. The emitted fluorescent signal (MFI) is measured using a Luminex 100 reader (settings: 50 events per bead, 50μl sample size, gate: 7500-13000 MFI).

Activation of antigen un-specific effector cells, such as NK cells results in tumour cell lysis (irrespectively on antigen expression)

Methods:

In vitro activation of human NK cells by immune serum

Human peripheral blood mononuclear cells (PBMCs) are isolated from buffy coats of healthy donors (obtained from the Austrian Red Cross) on a Ficoll density gradient. The NK cell enriched fraction is obtained from the non-adherent PBMC fraction (after removal of adherent cells by 1 h plastic adherence) and by negative sorting on an AutoMACS ™ using antibodies against CD3, CD 14, CD34, and CD 19. The NK cell enriched population is characterized by FACS analysis using CD56 / CD 16 as marker and consisted of at least 60 % NK cells. The NK-enriched cell fraction is incubated with pre-serum or immune serum (at a final dilution of 1 :5), and in the presence or absence of supplements of indicated amounts of hIL-2, for 48 h. The medium is replaced with fresh RPMI 1640 containing 10 % FCS and incubation is continued for another 24 h. After ishing the cells are re-suspended in fresh medium and used as effector cells in a 4 h ⁵¹ Cr-release lysis assay against labeled KATOIII tumour target cells at effector to target (E:T) ratios of 10:1 and 1:1, respectively. Release of chromium from lysed target cells into the supernatant of the samples ("Cs") is measured using a γ-counter (Cobra 5005, Canberra- Packard, Australia). Spontaneous release ("Sr") and maximum release (100 %, "Mr") are measured after incubation of target cells with medium alone or with detergent (2 % SDS), respectively. Cytotoxicity is calculated using the formula 100 % x (Cs-Sr) / (Mr-Sr).

Ex vivo measurement of activation of non-adherent PBMCs derived from Rhesus monkeys before and after immunization

PBMCs are isolated from heparinized blood derived from Rhesus monkeys before or after immunization, respectively, on a Ficoll density gradient. After washing, the non-adherent PBMC fraction is obtained by removal of adherent cells following 2 h plastic adherence. Non-adherent PBMC are incubated in RPMI 1640 supplemented with 10 % FCS for 2 h and subsequently used

as effector cells in an overnight ⁵¹ Cr-release lysis assay with labeled KATOIII target cells at the E:T ratios of 60:1, 30:1, 10:1 and 3:1. Cytotoxicity is calculated as described above.

Results:

In vitro activation of human NK activity by pre-incubation with immune serum

To elucidate whether the induced cytokines may have triggered further biological effects, the potential activation of NK cells is investigated. Natural killer (NK) cells are known to kill target cells in a MHC independent manner and without re-exposure to antigen. NK cells have been shown to be activated by cytokines such as IL-I, IFNγand IL-2 and can be induced by high dose IL-2 to obtain feature of Lymphokine-Activated Killer (LAK) cells. Our preliminary experiments using enriched human NK cells showed efficient lysis of the NK sensitive target cells K562, but only marginal efficacy against the NK resistant DAUDI cells or KATOIII tumour cells. In contrast, pre-incubation of the NK cells with high doses of recombinant IL-2 (1000 U/ml) resulted in pronounced lysis of DAUDI cells and KATOIII cells indicating the generation of LAK cells (data not shown). To test the effect of released cytokines on NK effector functions, the NK enriched cell fraction of human PBMCs is incubated for two days with 1 :5 diluted pre-serum or immune serum derived from Rhesus monkeys following repeated boost immunizations (at time points of the highest measured cytokine levels). In order to compensate for potential inhibitory factors present in the Rhesus monkey serum, pre-serum and immune serum are used either alone or supplemented with a moderate IL-2 dose (100 U/ml) which by itself is not sufficient to induce sufficient activation of NK cells to lyse DAUDI or KATOIII tumour cells (data not shown). Incubation of effector cells with immune serum is expected to enhanc the lytic activity of NK enriched human PBMCs against KATOIII target cells.

Rhesus monkey derived PBMCs show enhanced cytolytic activity against tumour cells after immunization

In order to test whether the in vitro activation of NK enriched PBMC by pre-incubation with immune serum is reflecting also a stimulation of cellular cytotoxicity in vivo, PBMCs derived from Rhesus monkey before and after immunization, respectively, are tested for their cytolytic effect on ⁵¹ Cr labeled KATOIII tumour cells at different E:T ratios. Before being used as effector cells the PBMCs are depleted of monocytes by plastic adherence and any Rhesus monkey derived serum is removed by repeated washing steps. It is expected that the cytolytic activity of

their PBMCs is enhanced following repeated immunization of the animals compared to the time points before immunization.

Example 15

Preparation of a recombinant mouse MMA383 antibody (rMMA383)

Methods:

Preparation of a molecular biological constructs for the expression of recombinant mouse

MMA383 antibody (rMMA383)

The bicistronic pIRES vector (Clontech Laboratories Inc., Palo Alto, USA) allows the expression of two genes on a high level and enables the translation of two consecutive open reading frames from the messenger RNA. In order to select positive transformants using a reporter gene, the internal ribosome entry site (IRES) is truncated in this expression vector, thus enabling lower expression rates to occur in this second reading frame. However, for our expression product, the original IRES sequence has to be re-established to enable our demands for the expression of the heavy and light antibody chains at nearly the same amount of expression to be met. DNA cloning and manipulations are carried out in accordance with standard methods (Maniatis et al.). Using PCR technology and the Advantage-HF PCR Kit (CLONTECH Laboratories Inc., Palo Alto, USA), the heavy and light chains of the MMA383 antibody are amplified. Firstly, primer sequences are used to introduce the desired restriction sites necessary for the insertion of the gene in the multi cloning site of pIRES, and secondly KOZAK sequences are inserted upstream of the open reading frames. The autologous signal sequences are used to direct the naked polypeptide chains into the secretory circulation.

After a transfection of the E. coli bacterium strain DH5alpha (Gibco-BRL), positive transformants are screened by PCR. The constructs are bi-directionally sequenced and used for further transfections of eukaryotic cells.

Transfection

The characterized eukaryotic strain, CHO (ATCC-CRL9096), is transfected with the above described expression vector construct. To this end, the DHFR selection marker is used in order to establish stable cell lines expressing rMMA383. In a 6- well cell culture plate, the cell line at with 4 raM L-glutamine to a content of 1.5 g/L sodium bicarbonate and showed upon

supplementation with 0.1 mM hypoxanthin and 0.016 mM thymidine, 90%; fetal bovine serum, 10% (Gibco-BRL). The cells are allowed to grow until a cell density of 50%. In the absence of serum, the cells are then transfected with 2 μg DNA according to the manufacturer's instructions, using Lipofectin reagent (Gibco-BRL). The transfection is stopped by the addition of complete medium after 6 or 24 hours.

Selection of Positive Transformants and Cultivation

Complete medium is replaced with selection medium 24 or 48 hours after transfection. The FCS in the complete medium is replaced with dialyzed FCS (Gibco-BRL, origin: South America). Positive transformants appeared as rapidly growing multi-cellular conglomerates 10 days after the selection. The concentration of rMMA383 is analyzed in the supernatants by specific sandwich ELISAs recognizing both the variable and the constant domains of the antibody. Those cells which showed high productivity are divided 1:10 and placed in 75 cm ² cell culture flasks for storage in liquid nitrogen. In parallel, these producers are subjected to rising selection pressures by adding methothrexate to the culture medium, and the cells are sowed in a 6- well cell culture plate. The method is repeated approximately two weeks later, when the cells had reached stable growth kinetics. Departing from a concentration of 0.005 mM, the MTX concentration is doubled at each selection circle until a final concentration of 1.28 mM MTX and, at the same time, subcultivation is effected in 96-well cell culture plates. The supernatants are assayed once a week by a specific sandwich ELISA which recognizes both the variable and the constant domains of the antibody. Stable cultures exhibiting the highest productivities are transferred into 75 cm ² cell culture flasks and stepwise transferred in 860 cm ² rolling cell culture flasks in nonselective medium. The supernatants are harvested, centrifuged, analyzed and subjected to further purification.

Analysis of Expression Products

The supernatants are assayed by a specific sandwich ELISA which recognizes both the variable and the constant domains of the antibody. The mAb IGN311 is coated with a concentration of 10 μg/ml on Maxisorp (NUNC) adsorption plates. The monoclonal IGN311 antibody recognizes the variable domain of the MMA383 antibody. The remaining active groups are blocked by incubation with 1% milk powder, and the supernatants are applied. The expressed antibodies are detected through their constant regions via rabbit anti-mouse IgGl-HRP conjugates (Biozym). The size determination of the expressed proteins is done by SDS polyacrylamide gel

electrophoresis using 4-14% acrylamide gradient gels in a No vex (Gibco-BRL) electrophoresis chamber. The proteins are silver-stained.

In order to immunologically detect the expressed antibodies, Western blots analysis is performed. The proteins separated by the SDS polyacrylamide gels are electrotransferred out onto nitrocellulose membranes (0.2 μm) using a Novex (Gibco-BRL) blotting chamber. The membranes are washed twice before the addition of the block solution (TBS + 3% milk powder BBL) and the antibody solution (10 μg/ml IGN311 antibody, mouse monoclonal anti-mouse IgG antibody (Zymed) or rabbit anti-mouse IgG γ-chain (Zymed) in TBS + 1% milk powder). Color development is performed using rabbit anti-mouse IgG-HRP or mouse anti-rabbit IgG-HRP conjugated antibody (BIO-RAD), diluted to 1:1000 in TBS + 1% milk powder, and an HRP color development reagent (BIO-RAD) is added according to the manufacturer's instructions. Isoelectric focusing gels are used to compare the purified expression products with the characterized murine MMA383 standard hybridoma antibody. The samples are loaded on IEF gels, pH 3-7 (Invitrogen), and the separation is carried out according to the manufacturer's instructions.

The proteins are visualized by silver-staining or immunological methods by means of Western blots. To this end, the proteins are loaded in a Tris-buffered SDS/urea/iodoactamide buffer and transferred onto nitrocellulose membranes. This is affected according to the same method as described for Western blots. The detection is performed with IGN311 anti-idiotypical antibodies.

Affinity Purification

A Pharmacia (Amersham Pharmacia Biotech) AKTA system is used. 1000 ml of clear culture supernatant containing the antibody are concentrated with a Pro-Varion 30 kDa cut-off (Millipore) concentrator, then diluted with PBS and packed on a 20 ml IGN311 Sepharose affinity gel XK26/20 column (Amersham Pharmacia Biotech). Contaminating proteins are removed by washing step with PBS+200 mM NaCl. The bound antibodies are eluted with 100 mM glycine, pH 2.9, and immediately neutralized with 0.5M NaHCθ3. The effluent is observed online at 215 nm and 280 nm and subjected to a subsequent HPLC analysis with a ZORBAX G- 250 (Agilent Technologies) column.

2000 ml of harvested supernatants from the roller bottle cultures are centrifuged, concentrated, diluted in PBS and purified to homogeneity by affinity chromatography using an IGN311 sepharose column. After elution, neutralization and dialysis against PBS, the final product is

measured by SEC-HPLC. A hybridoma-derived murine immunoglobulin standard of the same isotype is compared with rMMA383.

A further characterization of the expression product is performed by reducing and non-reducing silver-stained SDS-PAGES and Western blots. The expression products are detected by the specific anti-idiotypical antibodies, IGN311, and visualized by an anti-human HRP-conjugated antibody.

Example 16

Expression of a Hybrid Immunogenic anti -idiotypic Antibody (imitating Lewis Y)

The recombinant IgG2a Lewis-Y antibody combines the anti-idiotypical Lewis-Y mimicking hypervariable region and the highly immunogenic mouse-IgG2a constant regions. The recombinant IgG2a Lewis-Y antibody immunotherapy increases the immunogenity of the original anti-idiotypic antibody IGN301 produced by a hybridoma eel and induces a strong immune response against Lewis-Y and/or EpCAM over-expressed or presented by epithelial tumour cells. This immune response leads to the lysis of tumour cells by complementary activation or to the prevention of cell-mediated metastasization.

Molecular biological constructs of the recombinant IgG2a Lewis-Y antibody are inserted in the polycistronic vector. The recombinant IgG2a Lewis-Y antibody is transiently expressed in HEK293 cells, after this calcium-phosphate co-precipitation was performed in a micro spin system in the presence of FCS. After purification by an anti-Lewis- Y affinity column and qualification of the expression product, the recombinant IgG2a Lewis-Y antibody is formulated on Al(0H)3 and used as a vaccine in Rhesus monkey immunization studies at four 500 μg doses. A high immunogenicity as compared to that of the original IGN301 vaccine is to be observed. The induced immune response of the IgG type is analyzed by ELISA and is expected to show immunization antigen, Lewis-Y and EpCAM, specificities.

Binding of immune sera of Rhesus monkeys to tumour cells is measured by cell surface staining using a FACScan (Becton Dickinson). The EpCAM and LewisY expressing KATOIII tumour cells are incubated with serum (diluted 1 :40 in PBS with 2% FCS) for 2h on ice. For detection a goat F(ab') ₂ anti-human IgG (H+L)-PE conjugate (1 :100, Immunotech, Marseille, France) is used. Pre- and immune sera are analyzed in parallel. Background binding of pre-sera is set at

10% positive cells for comparison with the cell binding measurable after incubation with the corresponding immune sera.

For the ex vivo measurement of activation of non-adherent PBMCs derived from Rhesus monkeys before and after immunization, PBMCs were isolated from heparinized blood derived from Rhesus monkeys before or after immunization, respectively, on a Ficoll density gradient. After washing, the non-adherent PBMC fraction was obtained by removal of adherent cells following 2 h plastic adherence. Non-adherent PBMC were incubated in RPMI 1640 supplemented with 10 % FCS for 2 h and subsequently used as effector cells in an overnight ⁵¹ Cr-release lysis assay with labeled KATOIII target cells at the E:T ratios of 60:1, 30:1, 10:1 and 3:1. Cytotoxicity was calculated as described above.