WO2003038439A2 - Functional identification of targets on tissues and cells - Google Patents

Abstract

The present invention relates to a method for the identification of disease-specific molecules of animal or human cells comprising (a) identifying body fluids from animals or humans not affected by said disease which are cytotoxic for animal or human cells carrying disease-specific molecules; (b) incubating fractions of said animal or human cells carrying disease specific molecules with cytotoxic body fluids from animals or humans not affected by said disease identified in step (a) and with said animal or human cells carrying disease specific molecules; (c) identifying fractions that interfere with the cytotoxicity of said body fluids towards said animal or human cells; and (d) optionally, further fractionating fractions that have tested positive in step (c) and repeating steps (b) and (c) until a homogenous fraction has been identified, said homogenous fraction representing disease-specific molecules; or (b') splitting said body fluids identified in step (a) into at least two portions and depleting one of said portions from cytotoxicity-mediating molecules; (c') subjecting molecules, from said animal or human cells carrying disease-specific molecules to separation techniques that allow an unambiguous identification of each molecule, (d') incubating said separated molecules alternatively with a portion of said body fluid not depleted from said cytotoxicity--mediating molecules and with a portion of said body fluid depleted from said cytotoxicity-mediating molecules; and (e') identifying molecules that are recognized by said portion of said body fluid not depleted from said cytotoxicity-mediating molecules but not by said portion of said body fluid depleted from said cytotoxicity-mediating molecules. The present invention further relates to the identification of inhibitors or antagonists of the molecule thus identified and to the modification of such inhibitors or antagonists to improve their pharmacological properties. Finally, the present invention relates in a most preferred embodiment to a method that includes the formulation of the inhibitor or antagonist identified or improved in a pharmaceutical composition. The approach described in accordance with the present invention may also be termed a theranostic approach.

Description

Functional Identification of Targets on Tissues and Cells

The present invention relates to a method for the identification of disease-specific molecules of animal or human cells comprising (a) identifying body fluids from animals or humans not affected by said disease which are cytotoxic for animal or human cells carrying disease-specific molecules; (b) incubating fractions of said animal or human cells carrying disease specific molecules with cytotoxic body fluids from animals or humans not affected by said disease identified in step (a) and with said animal or human cells carrying disease specific molecules; (c) identifying fractions that interfere with the cytotoxicity of said body fluids towards said animal or human cells; and (d) optionally, further fractionating fractions that have tested positive in step (c) and repeating steps (b) and (c) until a homogenous fraction has been identified, said homogenous fraction representing disease-specific molecules; or (b') splitting said body fluids identified in step (a) into at least two portions and depleting one of said portions from cytotoxicity-mediating molecules; (c') subjecting molecules, from said animal or human cells carrying disease-specific molecules to separation techniques that allow an unambiguous identification of each molecule, (d') incubating said separated molecules alternatively with a portion of said body fluid not depleted from said cytotoxicity- mediating molecules and with a portion of said body fluid depleted from said cytotoxicity-mediating molecules; and (e') identifying molecules that are recognized by said portion of said body fluid not depleted from said cytotoxicity-mediating molecules but not by said portion of said body fluid depleted from said cytotoxicity-mediating molecules. The present invention further relates to the identification of inhibitors or antagonists of the molecule thus identified and to the modification of such inhibitors or antagonists to improve their pharmacological properties. Finally, the present invention relates in a most preferred embodiment to a method that includes the formulation of the inhibitor or antagonist identified or improved in a pharmaceutical composition. The approach described in accordance with the present invention may also be termed a theranostic approach. In the specification, a variety of documents is referred to. The disclosure content of these documents, including manufacturers' manuals, is herewith incorporated by reference in its entirety.

The search for cell specific target molecules is presently carried out predominantly by the systematic investigation using genomics and proteomics techniques (Siegel et al., Oncology, (1986), 1211 - 1231 ; Berthold et al., Human Neuroblastoma . -Recent Advances in Clinical and Genetic Analysis, (1993), XIII - XVI; Beckwith et al., Am. J. Pathol 43, (1993), 1089 - 1104; Brodeur et al., Cancer Metastasis Rev. 10, (1991 ), 321 - 333; Hanada et al., Cancer Res. 53, (1993), 4978 - 4986).

In most cases, the DNA-, RNA- and/or protein composition of normal and diseased tissue is quantified and compared. Potential target molecules can then be detected in diseased tissue on the basis of their overexpression. This approach, however, does not provide any hint to the function of the identified targets. In order to clarify whether the identified targets are at all useful for the development of new pharmaceutically active compounds, the function and compatibility for humans has to be subsequently investigated in rather time-consuming and expensive assays.

Recently, it has been shown that sera from healthy and diseased donors contain components of the immunoglobulin class that detect disease-specific antigens such as tumor-specific antigens.

Schmitt et al. (Klinische Padiatrie, 211 (1999), 314-318) describe that sera of healthy individuals contain IgM cytotoxic for tumor cells whereas neuroblastoma (NB) patients do not or only rarely contain such antibodies. They further show that injections with cytotoxic IgM lead to tumor arrest. The assay for cytotoxicity is carried out in that NB cell lines are cultivated and then complement dependent lysis of various sera from healthy donors is measured. Viability is assessed using propidium iodide. For the improvement of the therapy approach the authors conclude that the identification of the antigen recognized by the anti-NB-lgM is a necessary prerequisite.

Misek et al. (WO99/00671 ) disclose the identification of cellular protein antigens to which patients with cancer or risk of cancer may develop autoantibodies. The protein antigens thus identified may, in turn, be used to detect the presence of serum antibodies in individuals wherein the presence of autoantibodies may be seen as a marker for the onset of a tumor development. The document further suggests the use of such antigens for immunizing individuals against tumors. Exemplary tumors are lung cancer and neuroblastoma. Specifically, the method comprises separating antigen-containing protein mixtures, for example from isolated cancer cells or subcellular protein fractions thereof, by two-dimensional gel electrophoresis followed by transfer of separated proteins onto a membrane. Specific antigens in the protein mixture are detected by treatment of the membrane with a patient' s serum followed by detection of specifically bound antibody by the use of a second labelled antibody which specifically binds to the first antibody. Separated protein antigens are considered disease specific antigens if they show prominence in the presence of sera suspected of harbouring autoantibodies compared to control sera.

EP-B1 0 234 122 (Hellstrom, et al.) describes the generation of antibodies directed against the ganglioside GD3 which mediates lysis of tumor cells via activation of serum complement or antibody-dependent cellular cytotoxicity. The antibodies produced are preferably monoclonal antibodies. The patient's own serum can be used as the source of complement. Hellstrom found that several antibodies of the IgGβ and lgG2_a class were able to mediate lysis of tumor cells only against surface glycolipid antigens but not against protein antigens on the surface of the same tumor cells.

Ollert et al, Proc. Natl. Acad. Sci. USA 93 (1996), 4498 - 4503 is an earlier paper of the group that published the paper Schmitt et al. referred to above. This document discloses that normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines. The antigen recognized by the IgM antibodies responsible for the cytotoxicity was identified to be a non-glycosylated 260-kDa protein. The antigen was detected by anti-NB positive normal human sera in Western blots wherein membrane extracts of a neuroblastoma cell line were analysed. Interestingly, the cytotoxic IgM did not detect cultured melanoma cells as well as a variety of other tumor cells derived from bone marrow, the lymphatic system, colon, pancreas, bone, skeletal muscle and kidney. The authors further conclude that some of the most abundant ganglioside structures could be excluded as dominant target epitopes. These include GD2 and GD3. They further exclude GM2 although GM2 has been found in the art to be expressed on TE-85 osteosarcoma cells and human fibroblasts. Whereas the above-recited art has provided approaches for employing serum-derived components for diagnostic and therapeutic purposes, these approaches still suffer from severe disadvantages. For example, the work underlying the group of Schmitt and colleagues being a follow-up publication of the paper by Ollert and colleagues state that the further advancement of their approach is hampered by the non-availability of the tumor-specific antigen. On the other hand, the work of Hellstrom and colleagues necessarily relies on the use of patient-derived serum and thus has necessarily only a narrow therapeutic application range. Overall, there is a need in the art to provide quick and reliable methods for the identification of validated disease-related targets that are useful in the generation of pharmaceutical compositions that may be used to treat, inter alia, diseases that have a bad prognosis such as cancer. The technical problem underlying the present invention was thus to provide means and methods for the identification of such disease-related targets.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a method for the identification of disease- specific molecules of animal or human cells comprising

(a) identifying body fluids from animals or humans not affected by said disease which are cytotoxic for animal or human cells carrying disease-specific molecules;

(b) incubating fractions of said animal or human cells carrying disease specific molecules with cytotoxic body fluids from animals or humans not affected by said disease identified in step (a) and with said animal or human cells carrying disease specific molecules;

(c) identifying fractions that interfere with the cytotoxicity of said body fluids towards said animal or human cells; and

(d) optionally, further fractionating fractions that have tested positive in step (c) and repeating steps (b) and (c) until a homogenous fraction has been identified, said homogenous fraction representing disease-specific molecules; or

(b') splitting said body fluids identified in step (a) into at least two portions and depleting one of said portions from cytotoxicity-mediating molecules; (c') subjecting molecules from said animal or human cells carrying disease-specific molecules to separation techniques that allow an unambiguous identification of each molecule;

(d') incubating said separated molecules alternatively with a portion of said body fluid not depleted from said cytotoxicity-mediating molecules and with a portion of said body fluid depleted from said cytotoxicity-mediating molecules; and

(e') identifying molecules that are recognized by said portion of said body fluid not depleted from said cytotoxicity-mediating molecules but not by said portion of said body fluid depleted from said cytotoxicity-mediating molecules.

In accordance with the present invention, the term "disease-specific molecules" relates to molecules that are solely or predominantly or to a higher degree (as compared to non-affected animals or humans) expressed or found in cells of an animal or human affected by a disease. For example, the disease-specific molecules may be solely or predominantly or to a higher degree expressed in cells of a diseased tissue. In another alternative, these molecules may be expressed in cells of a body fluid. An example of the latter embodiment is a molecule that is specifically or predominantly or to a higher degree expressed on leukemic cells or on lymphoma cells. Whereas the disease-related molecules may only be expressed on the diseased cells, they may also be expressed in normal cells, albeit to a lower degree. The disease-specific molecules are preferably exposed on the surface of the cells. Alternatively, they may be expressed in the cytoplasm, in organelles of the cell or in the nucleus. The disease-specific molecules are, as a rule, of organic nature and, as a rule, furthermore high molecular weight compounds such as (poly)peptides (i.e. peptides comprising up to 30 amino acids or polypeptides comprising 31 or more amino acids) glycoproteins, carbohydrates, lipids, nucleic acid, to name some options. The disease-specific molecules may have a causative relation with the disease or they may be phenotypically but not in a causative manner linked with the disease (bystander effect).

The term „body fluid" refers to body fluids as obtained from an animal or human or fractions of said body fluids. Fractions of said body fluids can be obtained as described herein elsewhere. The term ..fractions [...] of said body fluids" relates to portions of body fluids that may be obtained by conventional methods and may be obtained as a result of size-fractionation of molecules contained in body fluids, fractionation on the basis of the electric charge of molecules contained in said body fluids, solid-liquid fractionations and so forth; see "Current Protocols in Protein Science", Wiley and Sons, eds. Coligan et al., current edition.

The term „animals or humans not effected by said disease" refers both to "healthy" animals or humans, i.e. animals or humans that by their own estimation, feel healthy or are phenotypically not diseased or to animals or humans that harbor a disease, but not the disease for which the disease-specific molecule is to be identified. If the animal or human harbors a disease, the disease should not be interrelated with (i.e. causive for or associated with, e.g., by way of enhanced or reduced expression levels) the disease for which a disease-specific molecule is to be identified.

The term „cytotoxic for animal or human cells" means, in accordance with the present invention, that the body fluids contain molecules that mediate cytotoxicity for animal or human cells carrying the disease-specific molecules. Whereas it is preferred that the body fluids comprise all molecules that are necessary to kill the animal or human cells, it is also envisaged that the body fluids may contain only a part of the machinery necessary to carry out the cytotoxic effect. For example, the present invention also envisages constellations where the body fluids contain antibodies that allow a complement-dependent lysis but are deficient in one or more molecules of the complement cascade. In such a case, it is necessary to add to the assay a further serum that contains the necessary components of the complement cascade or to add the missing complement components separately. Whereas the body fluids may not contain the components necessary to complete the killing of the animal or human cells, it is preferably envisaged by the present invention that antibodies involved in said killing are contained in said body fluids.

The term ..homogenous fraction representing disease-specific molecules" refers to a fraction that only or essentially contains these disease-specific molecules. "Essentially contains" means that at least 90%, preferably at least 95%, more preferably at least 98% and most preferably at least 99% such as at least 99.8% or 100% of the (high molecular weight) components of the fraction are represented by these and most preferably one disease-specific molecule(s). Naturally, this fraction usually also contains low molecular weight components such as buffer components, water etc. which are disregarded for the purpose of defining the homogeneity of the fraction.

The "splitting" [of] said body fluids" is effected in its simplest form by dividing a body fluid obtained in two or more portions, for example, by pipetting a certain volume of the body fluid into a second (or third) container.

It is not necessary that the splitting of body fluids results in two or more equally-sized portions. For example, it is usually envisaged that the portion not to be depleted from cytotoxicity-mediating molecules represents a rather small portion of the overall amount of body fluid available. This is because the portion of the body fluid employed in the depletion process may be subjected to the (various) steps in said depletion process one or more times. This would normally result in a loss of a part of the body fluid in each step of the depletion process.

The steps recited in items (c') to (e') are per se conventional steps well-established in the art of molecular biology. How to perform these steps in a standard fashion is well documented in text books such as Sambrook et al., "Molecular Cloning, a Laboratory Manual" CSH Press 1989, Cold Spring Harbor. Step (d') requires that the separated molecules are incubated both with portions of body fluids depleted and with body fluids not depleted from cytotoxicity-mediating molecules. Insofar, the term "alternatively" is intended to mean that the same batch of separated molecules is not incubated with the two types of portions of body fluid at the same time.

As compared to the prior art methods, the method of the invention particularly relies on the following advantages:

The source of the body fluids used in the target search is not limited to patient-derived sera. Further, the identification step of cytotoxic body fluids can be carried out as a high- throughput assay. Since live (adherent) cells are assayed for the presence of target molecules interacting with said body fluids and leading to the lysis of the target cells, the identification steps directly provide a functional link between target molecule and cytotoxicity. Accordingly, only those body fluids will be further analyzed that have proven in the first step of the method to provide this functional linkage. An additional advantage of the method of the invention is that it allows the direct identification of the target molecules either by well established techniques such as 2D gel electrophoresis combined with Western blot analysis or, for example by the, optionally repeated, fractionation of cellular components until a homogenous population of molecules has been derived. Other separation and immobilization techniques may alternatively be employed. The method of the invention thus allows the differentiation between unspecific and specific targets whereas only the latter are useful in the development of validated and effective pharmaceutically active compounds.

The above-referenced steps of the method of the invention may be characterized in detail in accordance with preferred embodiments of the invention as follows: The identification of body fluids from animals or humans not affected by a disease which are cytotoxic for animal or human cells carrying disease-specific molecules is referred to, in a specific embodiment of this invention, as Cell death inducing Substance Screening (CISS-assay). For example, cells from a diseased tissue or body fluid may be cultivated on microtiter plates and incubated with body fluids (or parts thereof) of different sources (for example, of different healthy humans or a combination of healthy humans and humans affected by an unrelated disease). Subsequently, the fluorescent dye propidium iodide is added to the cells resulting in a selective staining of the DNA of dead cells. The resulting changes in fluorescence intensity are measured in a plate fluorimeter and thus allow the identification of body fluids (or parts thereof) having cytotoxic activity against cells. These cytotoxic body fluids (or parts thereof) can now be employed in the search of disease-specific targets.

The purification of target-specific molecules (proteins) on cells is preferably effected by the inhibition of Cell death inducing Substances-assay (ICIS-assay).

For example, cultivated disease-associated cells are lysed and the protein components obtained are fractionated using chromatographic separation methods. The further elucidation of potential targets is effected using the ICIS-assay. In this process, the further purified protein fractions are incubated with the body fluids (or parts thereof) identified in the CISS-assay to be cytotoxic. Further, they are checked with regard to their capability to inhibit the cytotoxicity in the CISS-assay. Using the ICIS-assay, those fractions are determined which are binding to the cytotoxic body fluids (or fractions thereof) and inhibit killing of the cells.

The identification of specific target molecules on cells may be effected by a comparative analysis of depleted sera and non-depleted sera using a Comparative identification of Tumor Targets-assay (CITT-assay).

For example, subsequent or alternatively to the ICIS-assay target antigens are identified on a comparative basis, e.g. by Western blot, FAR-Western blot, etc.. Specifically, cytotoxic body fluids (or fractions thereof) are depleted of toxicity mediating molecules by repeated absorption to the cells or inhibiting fractions thereof that were determined in the ICIS-assay. After separation of all cellular proteins using preferably two-dimensional gelelectrophoresis potential target antigens may now be discovered using the following comparative approach:

The targets of the cytotoxic sera leading to the killing of disease-related cells are selectively identified upon absorption with non-depleted cytotoxic body fluids only (or fractions thereof) while non-specific cellular compounds are recognized by depleted as well as non-depleted body fluids (or fractions thereof).

The targets selected using the ICIS- and CITT-analysis may be subsequently further characterized using, in a preferred embodiment, mass spectrometry. For example, if a protein was identified, this protein may in recombinant or native form be used as the single target in the ICIS-assay in order to be finally confirmed as a target for the downstream molecular medicinal use (for example as a target for therapeutic or diagnostic immunoconjugates). In other terms, targets thus identified may be employed not only in the selection of inhibitors or antagonists (or even agonists) that, in turn, may form the basis of (the development of) a pharmaceutical composition. Rather, they may also be used for the immunization of individuals that may thus be in a position to develop a protective immune response to the target antigen. The immunization protocol may include a primary and one or more booster immunizations. Such a protective immune response may guard against the onset of a variety of diseases, in particular those that have a bad prognosis such as cancer. The above referenced immunization would be essentially only applied against antigens that are solely or essentially solely expressed on diseased cells in order to avoid the induction of an auto-immune response.

Advantageously, the method of the invention is carried out using biological sources such as cells and body fluids, from one species only. It is also preferred that all steps are carried out in vitro.

The disease-specific molecules may be derived or associated with a variety of tissues such as neuronal tissues or organ tissues. In a preferred embodiment of the method of the invention said disease-specific molecules are tumor-specific. In further preferred embodiments of the method of the invention, said disease-specific molecules are associated with or causative for allergy, infectious diseases, viral infections (e.g. HIV infections), cardiovascular diseases, neurodegenerative diseases (e.g. multiple sclerosis, Alzheimers disease), immune- and autoimmune diseases (e.g. rheumatic diseases, psoriasis), chronic fatique syndrom, or transplant rejection.

As has been demonstrated in the appended examples, the proof-of-principle of the method of the invention was obtained with the use of tumor cells. Given the fact that in particular malignant tumors in humans even today still have in many instances a rather bad prognosis, the present invention is expected to significantly contribute to the development of effective anti-tumor drugs. In this regard, it is important to note that the options conferred by the method of the invention are not confined to the tumors discussed in the examples, but to virtually any tumor carrying disease-specific antigens which allow the generation of cytotoxicity-mediating molecules, in particular antibodies, in body fluids of animals or humans.

In a further preferred embodiment of the method of the present invention said animal cells are mammalian cells.

Body fluids in accordance with the invention comprise any body fluid, such as urine, spinal fluids, saliva etc. It is also preferred in accordance with the method of the invention that said body fluids are sera, blood, and lymph or fractions thereof. An example of a fraction of blood is plasma. The method of the invention in another preferred embodiment requires that said body fluids are body fluids from humans.

Whereas the body fluid used in the validation of the target, may, in principle, also be derived from animals such as sharks, rabbits, cows, sheep, goats, horses or mice, it is particularly convenient if said body fluids are deπved from humans. The particular advantage of this embodiment resides in the facts that the direct confirmation of the effectiveness in humans is possible and no or essentially no side effects in humans will be observed.

The tumor-specific molecules may be derived from a variety of tumors. These include solid tumors, such derived from various different entities such as, breast, colon, lung, pancreas, head-neck, kidney, gastro-intestine, prostate, ovary, skin, liver testis, oesophagus, prostata, bone, bladder and brain, lymphomas such as Hodgkins- lymphoma, all kinds of non-Hodgkins-lymphoma, leukemias, such as all kinds of acute leukemia, all kinds of chronic leukemia, myeloproliferative syndromes, metastases, melanomas, rare tumors of unknown origin such as CUP, schwanoma, etc, of children with low frequency tumors, such as Ewing sarcoma (including Askin-tumor and peripheral neuroectodermal tumor), neuroblastoma , ganglioneuroma, osteosarcoma, Non-Hodgkins-Lymphoma, malignant teratoma, primitive neuroectodermal tumor, ependymoma, medulloblastoma, astrocytoma, rhabdomyosarcoma , synovial sarcoma, malignant fibrous histiocytoma, nephroblastoma etc. of adults with low frequency or with difficulty in clinical evalution due to low frequency of cases; in addition tumors specifically responsive to immune therapy, tumors which did not respond to conventional therapy, tumors with low response rates in terms of therapy, tumors not accessible to surgical interventions, tumors in patients not responding to vaccination therapy. Whereas this list is exemplary and comprises preferred embodiments, it is not exhaustive.

It is particularly preferred in accordance with the present invention, that tumor-specific molecules are molecules derived from adenocarcinoma, neuroblastoma, Ewing sarcoma. The molecules separated in accordance with the invention and comprising the target to be identified may be receptors, cell surface molecules etc. The targets may be of known or unknown origin.

According to a preferred embodiment of the method of the invention the molecules subjected to separation techniques are proteins, DNA molecules, RNA molecules, carbohydrates, gangliosides, lipids, glycolipids, steroids, lectins or peptides. The proteins or peptides may be modified such as by phosphorylation, farnesylation or glycosylation. The proteins may further be cell membrane proteins such as peripheral or integrated membrane proteins. They may, for example, be enzymes such as kinases. Receptors include hormone receptors (e.g. androgene receptors, estrogene receptors, corticoid receptors, progesterone receptors, testosterone receptor etc);neurotransmitter receptors (e.g. acetylcholine receptor, GABA receptor); cytokine receptors; peptide receptors; growth hormone receptors; e.g.: epithelial growth factor receptor, platelet derived growth factor receptor, vaso-endothelial growth factor receptor, nerve growth factor receptor, fibroblast growth factor receptor, GPCR, etc. The molecules may also be ion channels; e.g. calcium channels, chloride channels or potassium channels water channels; membrane transporters, or other molecules including e.g. cell adhesion molecules, E-cadherin, l-CAM, stroma, integrin or gap-junctions.

It is of note that after the completion of the separation step such as 2D-gel electrophoresis, the proteins may, in a preferred embodiment be transferred to one or two membranes and affixed thereon. If two membranes are employed, they bear an identical protein pattern. This alternative is convenient since in the following assay steps, one membrane can be contacted with the depleted body fluid whereas the second membrane can be contacted with the non-depleted body fluid. If, alternatively, the proteins are transferred to only one membrane, then the membrane has to be "washed" after development of the test with the depleted or non-depleted body fluid. After removal of the antibodies from the body fluid (depleted or non-depleted) used in the first contact with the membrane, the membrane may be contacted again with the second body fluid. The sample principle, of course, applies to other immobilization devices as well. In a more preferred embodiment of the method of the invention the separation techniques are selected from one or two dimensional gel-electrophoresis and overlay high performance thin layer chromatography (HPTLC).

According to a more preferred embodiment the molecules subjected to separation techniques are subsequently immobilized. More preferably said immobilization is effected by Western blotting, FAR Western blotting or TLC, (HPTLC) overlay techniques.

It is most preferred in accordance with the present invention that the molecules subjected to separation techniques are proteins or gangliosides, that the separation technique employed is 2D- gel electrophoresis, and that the immobilization technique employed is Western blotting or FAR Western blotting. The person skilled in the art is, without further ado, in the position to select the appropriate separation and immobilization techniques or any of the above referenced molecules subjected to separation.

The fractions of said animal or human cells carrying disease specific molecules may chemically be rather heterogeneous and comprise, for example, gangliosides, lipids, glycolipids, steroids, lectins or carbohydrates and nucleic acids in addition to proteins, or any of other types of molecules referred to above, depending on the fractionation protocol involved. Thus, and as stated elsewhere in this specification, fractionation may be effected on the basis of size, electrical charge, by affinity chromatography etc. cellular components may be divided into cytoplasmic components and nuclear components and fractionated accordingly. In another alternative, the fractions of said animal or human cells carrying disease specific molecules may be chemically rather homogeneous and comprise solely or predominantly e.g. carbohydrates or proteins, or gangliosides or nucleic acids. In an additionally preferred embodiment, the present invention relates to a method wherein fractions of said mammalian cells carrying disease specific molecules are protein fractions, ganglioside containing fractions, DNA, RNA or carbohydrate fractions. Also preferred in accordance with the method of the present invention is that said fractions of said animal or human cells carrying disease specific molecules are membrane fractions, nuclear fractions or cytosolic fractions.

In general, fractionation of course does not necessarily require the employment of chromatographic methods. Rather, the skilled artisan will choose an appropriate fractionation protocol on the basis of the molecule type involved taking also into account the potential or actual inhibitors contained in the fractions. For example, proteins may be separated from other cellular components by fractionation with ammonium sulphate. Nucleic acids, in particular DNA, may be purified from further cellular components using precipitation with alcohol. Yet, the method of the invention requires in a further preferred embodiment that said fractions obtained in step (c) or step (d) are obtained by chromatographic methods. In this regard, it is particularly preferred that size fractionation or fractionation on the basis of the electrical charge of cellular molecules is effected. Also possible is chromatographic fractionation on the basis of affinity employing, for example, antibodies or fragments or derivatives thereof.

The method of the invention requires in an additionally preferred embodiment that said homogenous fraction identified in step (d) consists of a single type of molecule. The identification of a single type of molecule will allow the direct employment of said molecule in the selection of antagonists or inhibitors or of agonists, for example useful in the development of vaccines. The compound may also be used in the further validation in diagnostic or therapeutic purposes. However, in principle, it is also possible that the homogeneous population of disease-related molecules comprises two or more disease- related molecules (but no molecules that are not related to the disease) that would normally and based on the selection process employed, have similar physico-chemical properties. Prior to the selection of antagonists or inhibitors (or agonists) it is envisaged to further separate these molecule to obtain a homogeneous fraction harboring only a single cellular compound.

It is also preferred in accordance with the present invention that the cytotoxicity- mediating molecules include antibodies. It is further preferred that the antibodies are capable of mediating ADCC or of activating the complement cascade. Most preferably, said antibodies are of the IgM type: In particular in the cases where the body fluid is serum, blood or lymph, antibodies, preferably of the IgM type are mediators of cytotoxicity. As mentioned above, cytotoxicity by serum components can be mediated essentially by two different mechanisms. The first mechanism is complement dependent lysis involving the activation of the complement cascade by the Fc-portion of antibodies and the second mechanism is antibody dependent cellular cytotoxicity (ADCC). Both mechanism are well known in the art and have been described, for example, in W.E. Paul (ed), "Fundamental Immunology" 2^nd edition, 1989, Raven Press, New York.

In accordance with the above, it is most preferred that said cytotoxicity-mediating molecules further comprise complement or cytotoxic T lymphocytes.

In another preferred embodiment of the method of the invention, said depletion in step (b') is effected by at least one round of incubation of the cytotoxic body fluid with said animal or human cells carrying disease-specific molecules.

Conveniently, the depletion step is repeated at least one more time. In any case, it needs to be tested whether the body fluid or fraction thereof still contains cytotoxicity- mediating molecules after a depletion round. Should this be the case, a further depletion round is required. Exemplarily, the serum is incubated with the mammalian cells carrying disease specific molecules and removed after having effected its detrimental effect. Antibodies as cytotoxicity-mediating molecules remain with the lysed cells. The remainder of the serum is again incubated with a new panel of live animal or human cells carrying disease specific molecules. If the serum still contains cytotoxicity- mediating molecules, dead cells will ensue. The procedure is repeated so many times until the incubation of the serum will no longer result in the lysis of said animal or human cells carrying disease specific molecules

In accordance with the above, it is also preferred that said disease-specific molecules are proteins, gangliosides, DNA, RNA, carbohydrates, lipids, glycolipids, steroids, lectins or peptides. The various molecule classes may carry the same modifications and may confer the same functions as has been outlined above for these types of molecules.

Generally, the molecules identified by the above referenced steps and preferably representing a single type of molecule may be further characterized by conventional methodology. For example, proteins may be sequenced employing standard methods such as mass spectrometry or Edman degradation. It this manner and if the molecules identified are proteins, the primary amino acid sequence may be determined. Alternatively and additionally, the proteins may purified to an extent that allows crystallization thereof. Successful crystallization of proteins or peptides in many instances requires merely conventional approaches such as described in Stevens RC, Curr. Opin. Struct. Biol., 2000, Oct;10(5):558-63.).Crystallization allows the identification of the binding pocket/ the active site of the protein interacting with natural receptors or with e.g. allosteric inhibitors. Crystallization may therefore be employed as one step in the identification of agonists, inhibitors or antagonists of the molecule identified in accordance with the invention.

Preferably, in accordance with the method of the present invention, said disease- specific molecule identified are further characterized by mass spectrometry (MS). However, other spectroscopic techniques may also be employed. MS has proven in recent years to be a reliable and quick method for the characterization of molecules. For example, proteins have been successfully characterized by mass spectrometry, as described in Mann M et al., Annu. Rev. Biochem., 2001 , 70, 437-73.

It is also preferred in accordance with the invention that said identification of body fluids in step (a) is effected by high-throughput screening (HTS).

HTS methods in accordance with the present invention are most preferably carried out in microtiter plates, conveniently in 96 well microtiter plates. Cytotoxicity may be detected, for example, in colorimetric assays, making use of the fact that the membrane of dead cells disintegrated and allows the inclusion of dyes. The amount of dye associated with dead cells may subsequently be determined in said colorimetric assay and, in turn, allow conclusions with respect to the degree of cytotoxicity of the body fluid tested. One preferred example of such a dye is propidium iodide. The color development per well may be determined using a conventional ELISA plate reader, optionally coupled to a CCD camera. It is most preferred in accordance with the present invention that said HTS is computer assisted.

The method of the present invention preferably further comprises identifying an agonist, inhibitor or antagonist of the disease-specific molecule identified. The agonist, inhibitor or antagonist identified is most preferably an antibody, peptide, aptamer or small molecule. In connection with the present invention, the term "antibody" also includes fragments or derivatives of an antibody. Fragments include Fab, F(ab₂)' and Fv fragments. Derivatives include scFv constructs, chimeric antibodies or humanized antibodies. The two latter embodiments include formats wherein a mouse V_H and V region have been grafted onto a human constant region and where CDRs from mouse antibodies conferring a desired binding specificity have been included into the framework region of a human antibody. Further guidance of how such fragments and derivatives may be generated may be found in the art, e.g. in Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor. Derivatives also include fully human antibodies produced e.g. in transgenic mice.

In a most preferred embodiment, the method comprises incubating said disease-specific molecule identified with one, several or a library of small molecules, peptides, aptamers or antibodies and identifying small molecules, peptides, aptamers or antibodies that interfere with or stimulate the biological activity of said disease-specific molecule or that interfere with or stimulate the binding of said disease-specific molecule with its receptor.

The agonist identified may be an activator of protein function or an activator of transcription or translation, to name a few examples. The agonist may be employed in the development or formulation of vaccines. The vaccines may be used for vaccination of the animal or human against a disease as identified elsewhere in this specification.

Additionally, the invention relates to a method comprising the further step of improvement or of refining the pharmacological properties of the identified, agonist, inhibitor or antagonist, by the method as described herein above, said method comprising the optionally the steps of said methods and:

(1) identification of the binding sites of the agonist, inhibitor or antagonist and the disease-specific molecule by site-directed mutagenesis or chimeric protein studies;

(2) molecular modeling of both the binding site of the agonist, inhibitor or antagonist and the binding site of the disease-specific molecule; and

(3) modification of the inhibitor or antagonist to improve its binding specificity for the disease-specific molecule. All techniques employed in the various steps of the method of the invention are conventional or can be derived by the person skilled in the art from conventional techniques without further ado. Thus, biological assays based on the herein identified nature of the disease-specific molecules may be employed to assess the specificity or potency of the drugs wherein the increase of one or more activities of the disease- specific molecules may be used to monitor said specificity or potency. Steps (1 ) and (2) can be carried out according to conventional protocols. A protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157- 178. The use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci. 61 :2507-2520. Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook (1989), loc. cit.. A fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway-system (Life technologies), a system that is based on DNA fusion by recombination. A prominent example of molecular modeling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251 -1265.

For example, identification of the binding site of said drug (i.e. agonist, inhibitor or antagonist) by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the (poly)peptide primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.

As regards step (2), inter alia, the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1 )) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the (poly)peptide of interest. Finally, in step (3) the drug can be modified to improve its binding affinity or its potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the (poly)peptide of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction.

Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative, agonist, inhibitor or antagonist and the disease-specific molecule, for example a (poly)peptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1 -13; Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of activators of the expression of a (poly)peptide as an example of a disease-specific molecule identified in accordance with the invention can be used for the design of peptidomimetic activators, e.g., in combination with the (poly)peptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

In accordance with the above, in a preferred embodiment of the method of the invention said pharmacological properties of the identified agonist, inhibitor or antagonist is further improved or refined by peptidomimetics.

The invention furthermore relates to a method of modifying an agonist, inhibitor or antagonist identified, improved or refined by the method as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico- chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl groups to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidinesor combinations thereof; said method optionally further comprising the steps of the above described methods.

The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

The invention moreover relates to a method of producing a pharmaceutical composition comprising optionally the steps of the aforementioned methods and further the step of formulating the agonist, inhibitor or antagonist identified, improved, refined or modified by the method of any of the preceding claims with a pharmaceutically active carrier or diluent.

The pharmaceutical composition produced in accordance with the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

In an additionally preferred embodiment, the method of the invention requires that the disease-specific molecule identified is the GM2 or the MHH-ES1 ganglioside. The above referenced molecules have been identified to be associated with Ewing sarcoma. Accordingly, these gangliosides can be employed in the various approaches referred to herein above to develop a cure for Ewing sarcoma.

The invention also relates to the use of a human serum or plasma derived from a healthy donor that comprises antibodies mediating cytotoxicity against cells harboring the GM2 or the MHH-ES1 ganglioside in the preparation of a pharmaceutical composition for the treatment of a malignancy. Appropriate malignancies have been identified herein above. Preferably, said malignancy is Ewing sarcoma. The formulation and constituents of a pharmaceutical composition to be prepared in accordance with the present invention have been discussed in detail herein above. Using this theranostic approach, humans suffering from Ewing sarcoma may be successfully treated since the antibodies are expected to at least reduce and optimally extinguish the tumor load. In general, cytotoxic sera against tumor cells or fractions thereof can be used for the therapy of patients suffering from the corresponding tumor. Sera or plasma toxic for the tumor can be applied by plasmapheresis or transfusion. Therapy with cytotoxic sera can be applied in combination with conventional therapy (i.e. chemo- and radiotherapy).

A further preferred embodiment of the method of the invention further comprises the step of formulating the disease-specific molecule identified as a vaccine. Said vaccine may further comprise conventional constituent such as adjuvants. In one embodiment, the molecule may be encased into liposomes or related molecules such as virosomes. Uses of said vaccine and vaccination schedules have been discussed herein above.

The various definitions, applications and further elaborations on the therapeutic and diagnostic options now possible in accordance with the invention as well as the further preferred embodiments described above apply mutatis mutandis to the following specific embodiments of the present invention. The invention in a particular embodiment relates to a method of identifying tumor- specific antigens on cancer cells characterized in that

(a) human sera with cytotoxic characteristics against cancer cells are identified;

(b) protein fractions from lysed cancer cells are incubated with these cytotoxic sera; (d ) those protein fractions that inhibit the killing of the cancer cells are identified and purified; or (c2) target antigens are identified via western blot analysis; and (d) subsequently a characterization is effected via mass spectrometry and/or via

Edman sequencing. In a preferred embodiment of the method of the invention, the identification of human sera with cytotoxic characteristics is effected by:

(a) incubation of tumor cells with sera of different origin;

(b) subsequent selective staining of the killed cells; and

(c) subsequent determination of the fluorescence.

It is also preferred in accordance with the present invention that the protein fractions are obtained from the lysed cancer cells by chromatographic separation techniques and a repeated purification is effected by the respective determination of the inhibition until a homogeneous molecule component is obtained.

It is also preferred in accordance with the method of the invention that the human sera with cytotoxic characteristics are separated from the toxic molecules by repeated adsorption to the cancer cells.

It is particularly preferred that the human sera with cytotoxic characteristics are separated from the toxic molecules by repeated adsorption to the cancer cells, that the separation of the total protein of the cancer cells is effected via gel-electrophoresis and that the identification of target-antigens is effected by comparative western blot analysis.

The invention also relates to the detection and use of body fluids displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

Further, the invention pertains to the detection and use of fractions of body fluids displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

Additionally, the invention relates to the detection and use of antibodies, antibody fractions or subclasses of antibodies from human blood serum or plasma displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

The invention further relates to the detection and use of body fluids or fractions thereof for the identification of specific targets which mediate cytotoxicity on malignant cells.

Another object of the invention is the use of targets detected in accordance with the method of the invention for the preparation of a therapeutic or diagnostic composition for the therapy or diagnosis of human malignancies.

Finally the invention relates to the use of body fluids and the FITT™ Technology for predictive assays in therapy.

The figures show:

Figure 1 : Schematic overview over the method of the invention for searching targets using cytotoxic body fluids (or fractions thereof).

Figure 2: Determination of the cytotoxicity after incubation of HT29 cells with five different sera (CISS-assay). For comparative purposes, the cytotoxicity of negative controls (FCS, foetal calf serum) is shown. The cytotoxic serum 2979 was employed for the subsequent analysis.

Figure 3: Determination of inhibition of cytotoxicity of serum 2979 by pre-incubation with protein fractions from HT29 cell extracts (ICIS-assay). Fractions 1 to 4 were obtained by gel filtration of HT29 cell extracts on sephacryl 300HR. A: positive control (serum 2979); B: negative control (non-toxic serum); C: buffer control. Fraction 1 shows the highest inhibitory effect. Figure 4: Comparative analysis of the cytotoxic effect of serum 2979 before and after depletion of cytotoxic components towards HT29 cells in the CISS-assay. As a negative control, FCS was employed.

Figure 5: Comparative Western blot analysis (CITT-assay) of depleted (B:) and non- depleted (A:) toxic serum 2979 on HT29 cell extracts. Black arrow: Protein-target band. In the depleted serum corresponding IgG antibody is missing due to absorption on HT29 cells. White arrow: unspecific band that does not represent a cytotoxic target and is recognized by native (non-depleted) and depleted serum.

Figure 6A: The cytotoxicities of different human sera against 3 different Ewing sarcoma cell lines (MHH_ES1 , SK-N-MC, CADO-ES1) are displayed. The 100% cytotoxicity value is determined at the end of the experiment by killing all cells in the well with 4% Triton X-100.

Figure 6B: Detection of anti-gangloside IgM antibodies in different donor sera. The different gangliosides were coated on microtiter plates (gangliosides purified from bovine brain, GT1 b, GD2, GM2 and GM1 ). Sera found to be toxic to the Ewing sarcoma cell lines (see in Fig. 6A) were tested. The non-toxic serum is the same as displayed in Fig. 6A. A cytotoxic control serum (■) was set to 100% as a reference according to its anti-GM2 IgM content.

Figure 7: ICIS-Assay on MHH-ES1 cells with the cytotoxic serum 2 (see Fig. 6A and 6B for cytotoxicity and IgM content, respectively). This serum was pre-incubated with GM1 , GD2, GM2, bovine brain gangliosides and ganglioside preparations from MHH-ES1 cells. The toxicity of this serum 2 against Ewing sarcoma cells can be specifically inhibited by MHH-ES1 ganglioside preparations and purified GM2. FCS and PBS are controls for residual cell death of MHH-ES1 cells.

Figure 8: Cytotoxicity of serum 2 after depletion on MHH-ES1 cells (center bar). Left bar: Cytotoxicity of non-depleted serum 2. Right bar: non-toxic control serum. The 100% cytotoxicity value is determined at the end of the experiment by killing all cells in the well with 4% Triton X-100. Figure 9: Anti-GM2 IgM antibody content of depleted and non-depleted serum 2. Center (black): depleted serum 2; Left bar: toxic serum 2; Right bar: non-toxic control serum. The antibody content of serum 2 was set to 100%.

The Examples illustrate the invention.

Example 1

Materials and Methods

Human body fluids (sera):

Sera from different patients/donors from Megamedics' own serum bank as well as of cooperating blood donor banks were tested.

Tumor cells:

The HT29 cell line (a colon adenocarcinoma cell line) was obtained from the American Type Culture Collection (ATCC HTB-38) and according to the published prescription cultivated and passaged.

High throughput-cvtotoxicity tests on microtiter plates (CISS-assay):

HT29 cells were transferred using a 12-channel pipette into 96-well-microtiter plates and cultivated at an initial density of 20,000 cells per well two days in 100 μl culture medium (McCoy's 5A in 10% FCS). After removal of the culture medium, the exponentially growing adherent cells were overlaid with 20 μl of the test sera, respectively, and incubated one hour at 37°C. Subsequently, 30 μl of propidium iodide (PI, 40 μg/ml in phosphate buffer) were added per well and incubated for an additional hour at 37°C. PI is a fluorescent dye that invades cells with disrupted cell membranes, then intercalates with DNA and exhibits a specific emission at 617 nm upon excitation with light of a wave-length of 536 nm. The fluorescence of cells killed by the toxic sera and stained with PI were measured in a platefluorimeter (BioTek FL600) so that the relative toxicity of the tested sera could be determined. As a negative control, cells were employed which were incubated with non-toxic culture serum (heat-inactivated FCS). As a positive control (100% PI insertion), cells were incubated in 50% dimethylsulfoxide (DMSO).To determine the percentage of cells killed by the cytotoxic serum, into the microtiterplates under estimation, 10 μl per well of a 20% triton-X-solution, comprising PI, 40 μg/ml in phosphate buffer) were added and incubated one hour at 37°C. This treatment lead to the permeabilization of the cell membrane and thus to the Pl-staining of all cells contained in the well. After the incubation, a second measurement is carried out in the platefluorimeter. Thus, the cell number of all cells contained in the well is determined and the percentage of the cells killed by the respective serum can be calculated.

Preparation of cell extracts:

HT29 cells grown in culture flasks were trypsinized, twice-washed with phosphate buffered saline, transferred to 50ml plastic flasks and frozen in the presence of phosphate buffered saline (PBS) with complete protease inhibitor. After thawing, the cells were centrifuged 15 minutes at 300g. The supernatant was disguarded and the cell pellet suspended in 5ml extraction buffer (PBS with Complete Protease Inhibitor, 0.04mg/ml DNasel, 0.02mg/ml RNaseA, 2 mM MgCI₂) and transferred to 2 ml reaction vials. The centrifugation and the re-suspension in extraction buffer were repeated twice. Subsequently, the cells were suspended in the same volume of extraction buffer. Lysis of the cells was effected using ultrasonic treatment (ultrasonic processor type UP200s, Dr. Hielscher; Setting: cycle 0.7, amplitude 80%). Per experiment, ten ultrasonic periods were employed. Afterwards, the extracts were centrifuged one hour at 15,000 g and 4°C. The supernatant was set with PBS to a protein concentration of 10mg/ml and subsequently employed ICIS-assay.

The chromatographic separation of protein extracted from HT29 cells was carried out on a gel filtration matrix (Sephacryl 300HR, Amersham Pharmacia). The column was coupled to a liquid chromatography device (BioCad-sprint, Applied Biosystems). The isocratic running buffer employed was PBS, the flow rate did not exceed 0.5ml/min. The eluate was detected at 214 and 280 nm and fractions of respectively 2ml were collected

ICIS-assay: The ICIS-assay was carried out, in principle, the same way as the CISS-assay described here above in microtiter plates. In this case, the cytotoxic serum identified in the CISS-assay is incubated at 37°C with the chromatographically separated protein fractions prior to the assay. In a comparison with corresponding controls both fractions are determined that interfere with the cytotoxicity of the toxic sera. Positive fractions can be employed in the subsequent CITT-assay.

Alternatively, for the measurement of the inhibitory activity of a cell extract or of a chromatographically purified fraction on to the serum-mediated cytotoxicity initially 100 μl of the test sample were incubated with 100 μl of a cytotoxic serum for 2 hours on ice. Subsequently, the mixture was centrifuged 10 min. at 4°C and 15.000 xg. The supernatant was removed and incubated with 100 μl of a suspension of HT29 cells (5 x 10⁶ cells/ml in PBS with 0.1% gelatine) for 1 hour on ice. Subsequently, 500 μl PBS with 0.1% gelatine were added, mixed well and the resulting mixtures centrifuged for 5 min. at 4°C and 190 xg. The supernatant was discarded and the pellet was resuspended in 500 μl PBS with 0.1% gelatine. A further centrifugation was effected for 5 min. at 4°C and 190 xg and the pellet was suspended in 100 μl PBS with 0.1% gelatine. After addition of 100 μl of a non-toxic serum, die mixture was incubated for 1 hour at 37°C. Subsequently, 500 μl PBS with 0.1% gelatine were added and centrifuged for 5 min. at 4°C and 190 xg. The supernatant was discarded and the pellet was suspended in 400 μl PBS with 0.1% gelatine. To the samples subsequently 3 μl PI (0.25 mg/ml in phosphate buffer) were added and the mixture analysed in the flow cytometer (FACS). As controls, a cytotoxic and a non-cytotoxic serum as well as a buffer solution were used wherein the sample volumina were adjusted by addition of PBS with 0.1% gelatine. The flow cytometer was set as follows: excitation 488 nm, emition 536 nm. Per probe 5000 cells were analysed and the results were interpreted with the software provided by the manufacturer.

Comparative analysis using depleted sera (CITT-assav):

For the depletion of the cytotoxicity of sera, 80% confluent HT29 cells (about 10⁷ cells) were washed with PBS and incubated with 10ml of the serum 2979 toxic for HT29 cells for 1 hour at 37°C. The serum-supernatant was subsequently transferred to a fresh HT29 cell culture and again cultivated for 1 hour at 37°C. The transfer and incubation steps were altogether carried out eight times. The cytotoxicity of the depleted sera were subsequently assayed in the CISS-assay and the complete depletion of the cytotoxicity was corroborated.

For the comparative Western blot-analysis, protein extracts of HT29 cells were separated via 1 D or 2D gelelectrophoresis and subsequently transferred to PVDF- membranes. For the identification of tumor cell antigens, in parallel-produced blots were incubated with native serum 2979 (not depleted) and depleted serum 2979, respectively. As the secondary antibody, human immunoglobulin coupled to Horse-Radish- peroxidase were employed. The detection was effected using a chemiluminescent substrate. Protein bands that appear selectively only on the Western blot developed with native serum 2979 represent potential tumor cell targets. The corresponding protein spots or bands were excised from the gels, enzymatically fragmented and the fragments thus obtained analyzed by mass-spectrometry.

Results:

Sera of different blood donors were analyzed in the CISS-assay with respect to their cytotoxic characteristics. Figure 2 shows that, as compared to the negative control (FCS), several sera induced a significant enhancement of the percentage of Pl-stained cells and are therefore cytotoxic towards HT29 cells. The CISS-assay thus allows a unequivocal identification of sera having cytotoxic activity for different cancer cell lines.

For the purification of target proteins, HT29 cells were lysed and the solubilized protein components were fractioned by gel filtration using sephacryl 300HR. The various fractions were subsequently assayed in the ICIS-assay with regard to their capability of inhibiting the cytotoxic activity of serum 2979. The cytotoxicity of the samples measured in the ICIS-assay is demonstrated in Figure 3. These results show that fraction 1 causes a strong inhibition of the cytotoxicity of serum 2979. Apparently, components of this fraction bind during the pre-incubation with serum 2979 the toxicity mediating serum components. This approach thus allows the identification of target molecules that mediate the cytotoxic effect of the serum. The protein fractions were now further purified using chromatographic separation methods in combination with a test in the ICIS-assay until finally a homogeneous and single target protein was obtained. For the identification of target antigens by the CITT-assay, cytotoxic sera were depleted by repeated absorption to cancer cells from their toxicity. Figure 4 shows that the cytotoxicity in the CISS-assay of the cytotoxic serum 2979 is drastically reduced after depletion of the serum. The depleted serum 2979 practically does not display any cytotoxic activity towards HT29 cells in the CISS-assay. After the separation of membrane fractions of whole protein from HT29 cells by gelelectrophoresis, potential target antigens can now be identified by comparative Western blot analysis in the CITT- assay, as shown in figure 5. The corresponding protein bands or spots are selectively only detectable in Western blot developed with non-depleted serum. In contrast, unspecific proteins are recognized by depleted as well as non-depleted serum. It is further shown in Figure 5 that the specific target bands or spots are enriched in the membrane fractions solubilized with various detergents wherein the membrane fraction corresponds to fraction 1 of Figure 3.

The tumor targets selected with the ICIS- and CITT-analysis are subsequently further characterized by bio-analytical techniques (e.g. mass spectrometry, NMR and/or Edman sequencing). The identified protein is then, recombinantly or in native form, used as a single antigen in the ICIS-assay in order to be finally validated as a target antigen for the further medicinal use (for example a target for therapeutic or diagnostic immunoconjugates).

Example 2

Identification of cytotoxic sera against human Ewing sarcoma cells and of the cellular targets of these sera.

In the present invention human sera from blood donors were used to screen for sera which were able to induce CDC (complement dependent cytotoxicity) on human Ewing sarcoma cell lines CADO-ES1 , MHH-ES1 and SK-N-MC. Several sera cytotoxic for Ewing sarcoma cells were identified. The cellular molecules capable of inhibiting the cytotoxic sera were found to be contained in the membrane fraction of Ewing sarcoma cells. The membrane fraction was subfractionated into protein and glycolipid fraction as described elsewhere (Current protocols in protein science, Wiley and Sons, Editors: Coligan et al.). There was significant inhibition of the cytotoxicity of the sera in the ICIS- assay (see material and methods) due to the glycolipid fraction. Therefore the glycolipid fraction was used for further investigations. Gangliosides are prominent components of the glycolipid fraction. Ganglioside extracts were prepared from cells and separated by HPTLC.

A toxic serum was depleted from its toxicity by repeated adsorption to the MHH-ES1 Ewing sarcoma cells. For the detection of anti-ganglioside antibodies HPTLC's of cellular gangliosides were incubated with the depleted and the non-depleted toxic serum and compared. The HPTLC of the Ewing sarcoma cells incubated with the non-depleted toxic serum displayed a specific spot which was absent in the HPTLC incubated with the depleted serum. This spot was identified to be GM2 by using purified GM2 as a reference in the HPTLC.

In order to confirm GM2 as the cellular target of the cytotoxic serum, the serum was tested by ELISA for residual IgM antibodies against different gangliosides. The direct responsibility of the residual anti-ganglioside IgM antibodies for the sera induced CDC was proven in the ICIS-assay by preincubation of the cytotoxic sera with purified gangliosides, gangliosides purified from bovine brain or ganglioside preparations from the Ewing sarcoma cells. Both the ganglioside preparations from the Ewing sarcoma cells and the purified GM2 were shown to inhibit the CDC.

The experiments demonstrate that toxic sera from healthy human donors can be identified. Furthermore proof is provided that these sera are competent to kill Ewing sarcoma cells due to their enhanced anti-GM2 IgM antibody content in comparison to serum depleted from its toxicity or non-toxic control serum.

Material und Methods:

Sera:

Different human sera from cooperating blood donation facilities were used for screening of cytotoxic effects on Ewing sarcoma cell lines. All donors signed an inform consent thereby agreeing the use of their sera in the experiments.

Tumor cell lines:

Ewing sarcoma cell lines MHH-ES1 (DSMZ No. ACC 167), SK-N-MC (DSMZ No. ACC 203) and CADO-ES1 (DSMZ No. ACC 255) were obtained from the „Deutsche Sammlung von Mikroorganismen und Zellkulturen" and cultivated according to the instructions of the supplier. Cytotoxicity test = CISS-Assay (Cell Death Inducing Substance Search):

Exponentially growing cells were trypsinised from culture flasks (Greiner) and plated on 96 well microplates (Greiner) at a density of 40.000 cells/well in 100 μl culture medium (GIBCO) containing 10% FBS (GIBCO) and incubated at 37°C with 5% C0₂ atmosphere for 24 hours. After removing the culture medium the exponentially growing cells were incubated with 20 μl/well of donor sera at 37°C with 5% C0₂ atmosphere for 1 hour. 20 μl propidium-iodide solution (PI, 40 μg/ml in PBS) per well was added und incubation was continued for another hour. PI is a fluorescence dye staining only cells with disrupted cell membranes. It intercalates into the nuclear DNA of dead cells and upon excitation with 536 nm light it shows emission at 617 nm. The Pl-fluorescence of cells incubated with donor sera was measured in a fluorimeter (BioTek FL600). As a negative control cells were incubated in parallel with culture serum (heat inactivated FBS). The total number of cells per well was determined on the basis of their Pl-fluorescence by killing all cells by addition of 10μl/well of a 20% Triton-X solution (v/v, containing 40 μg/ml PI in PBS) and incubation at 37°C for one hour.

This treatment leads to the permeabilisation of the cell membrane and accordingly to PI fluorescence by all of the cells contained in the well. This procedure is used to determine the 100% toxicity value. The percentage of cells killed by the cytotoxic donor sera is calculated according to the fluorescence intensity of cells/well killed by donor sera. Experiments were performed in triplicates.

Preparation of ganglioside extracts:

1 ,5x10⁸ frozen cells were thawed on ice and pelleted at 3.000g for 5 min at 4°C (in a

Sigma Laboratory Centrifuge Typ GK15). The pellet was resuspended in 2 ml 80% (v/v) tetrahydrofuran (Merck 1.08110) in water (Millipore, Direct-Q) with a 1 ml pipette for 2 min, vortexed for 30 s and sonificated with 4x20 pulses on ice (cycle: 0,5 amplitude

50%, UP 200s, Dr. Hielscher GmbH).

The suspension was distributed to two 2 ml Eppendorf tubes and centrifuged at 6000 g for 10 min at 20°C (Eppendorf centrifuge 5417 R) . The supernatants containing the lipid fraction were transferred to new Eppendorf tubes.

This procedure was repeated twice with the remaining pellet. The tubes containing the supernatants were mixed with 0.3 volume diethylether (Merck 1.00930) using a 1 ml pipette, vortexed for 30 s (20°C), centrifuged at 4.000 g for 5 min at 20°C (Eppendorf centrifuge 5417 R). The upper phase and the inter phase (containing proteins) were carefully removed and discarded. The lower phase containing the gangliosides was vortexed for 30 s (at 20°C) and centrifuged at 4.000 g for 5 min again (at 20°C in an Eppendorf centrifuge 5417 R) to ensure its homogeneity. The remaining upper phase was carefully removed and discarded. The lower phase contained the gangliosides and was pooled and filled up with water to a total volume of 3 ml (Millipore, Direct-Q). 300 μl aliquots of this solution (containing the gangliosides of 1 ,5x10⁷ cells) were distributed to 10 Eppendorf tubes.

The aliquots were dried in the speedvac (Savant Speedvac plus SC210A, Thermo Quest) for 3h at RT and stored at -20°C for further experiments.

Overlay high performance thin layer chromatography (HPTLC):

HPTLC was performed as described elsewhere (Mϋthing et al., J. of Chromatogrophy A, 720 (1996), 3-25; Nobile Orazio et al., Ann. Neurol. 28 (1990), 190-194). In brief, ganglioside extracts from Ewing sarcoma cells were separated in parallel on silica gel HPTLC plates (MERCK, Germany) and incubated with depleted or non- depleted sera. Purified gangliosides (SIGMA) or gangliosides prepared from bovine brain (Type III, SIGMA) were used as a reference. Antibody binding was detected using anti-human IgG or IgM HRP conjugate (SIGMA) with Super signal (PIERCE) as a substrate.

Comparative analysis with depleted sera = CITT-assay (Comparative identification of tumor targets) using the overlay HPTLC:

The cytotoxicity of toxic sera was depleted using MHH-ES1 cells. An 80% confluent culture was incubated with 20 ml aliquots of cytotoxic serum (30 min at 37°C) and afterwards this serum was transferred to another 80% confluent culture. This transfer and incubation step was repeated eight times with fresh cell cultures. The reduction of the cytotoxicity of the depleted serum was confirmed in the CISS-assay against the same cytotoxic serum which was not depleted.

The absence of cytotoxic antibodies against GM2 in the depleted serum compared to the non-depleted serum was confirmed by the ganglioside ELISA (see below). Coating of 96 well plates with gangliosides:

DNA-BIND 96 well N-Oxysuccinimide Surface Amine Binding Polystyrene plates (Costar 2505) were used for the assay.

1μg of gangliosides GM1 , GM2, GT1 b or GD2 (SIGMA, CALBIOCHEM) or gangliosides purified from bovine brain (Type III, SIGMA) in 100 μl PBS were coated onto the plates respectively. For background controls 1μg BSA was coated in 100 μl PBS onto three wells of the plate. Incubation occurred at 4°C in the dark for 16h. The ganglioside coated plate was washed three times for 5min with 200μl 1 % BSA. Blocking occurred overnight with 200 μl 1% BSA per well. The supernatant was discarded and the plates stored at 4°C in the dark.

ELISA for the determination of anti-ganglioside antibodies in human sera:

Sera were added in a 1 :50 dilution (100 μl / well) into the ganglioside coated wells. Incubation occurred at 4°C in the dark over night. The plate was washed six times for 5 min with 200 μl 1 % BSA in PBS (w/v). Then the secondary antibody ahlgM (anti human IgM-HRP conjugate, 1 :10.000) in 1% BSA/PBS (100μl/well) was incubated at RT for two hours. The plate was washed six times for 5min with 200μl 1 % BSA/PBS. Detection occurred with OPD (o-phenylenediamine): Substrate was prepared 10 min before use as follows : 15 mg OPD was dissolved in 15ml 0,1% sodium citrate (pH 4,5) and 12μl H₂0₂ was supplemented immediately before use. 100 μl/well of this solution was added to each well followed by incubation at RT for 20 min in the dark. Before measurement 100μl 3 M HCl was added to each well and absorption was determined at 460 nm in the microplate reader. Experiments were performed in triplicates.

Inhibition assay = ICIS-assay (Inhibition of Cell death Inducing Serum):

The ICIS-assay was performed on culture cells in 96-well microplates. In this assay the cytotoxic serum identified in the CISS-assay was pre-incubated for 1 h at 37°C with purified gangliosides (SIGMA, CALBIOCHEM), gangliosides purified from bovine brain (Type III, SIGMA) or ganglioside extracts (see above) prepared from the MHH-ES1 cells. In this way gangliosides were identified which are able to inhibit the cytotoxicity of the sera. After removing the culture medium, the MHH-ES1 cells were incubated with 20 μl of the serum/ganglioside sample for 1 hour. All subsequent steps were identical to the CISS-assay (see above). Results:

Sera from different healthy donors were analyzed in the CISS-assay with regard to their cytotoxicity against three different Ewing sarcoma cell lines. Fig. 6A shows exemplary, that in comparison to the average cytotoxicity of human sera (negative control = non- toxic serum) specific sera (cytotoxic serum 1 and 2) induce a significantly higher PI fluorescence, thereby representing a high cytotoxicity. The CISS-assay allows the identification of these sera that are cytotoxic against Ewing sarcoma cell lines.

The cytotoxic sera identified in the CISS-assay were analyzed in the ganglioside ELISA with regard to their anti-ganglioside antibody content. The cytotoxic sera 1 and 2 contained a significantly higher amount of anti-ganglioside IgM antibodies compared with the non-cytotoxic human sera (Fig. 6B).

The serum 2 which is cytotoxic against MHH-ES1 Ewing sarcoma cells, was pre- incubated with purified GM2, GM1 , GD2, gangliosides purified from bovine brain and gangliosides purified from MHH-ES1 cells and analyzed in the ICIS-assay for the inhibition of the cytotoxicity of serum 2. Only GM2 and gangliosides purified from MHH- ES1 cells were capable of inhibiting the toxicity of this serum against the Ewing sarcoma cell line MHH-ES1 (Fig. 7).

In a further experiment serum 2 was adsorbed on MHH-ES1 cells in order to deplete the toxic compounds. The cytotoxic activity of the depleted serum 2 was reduced to the level of a non-toxic control serum (Fig. 8).

The depleted serum 2 was compared to the non-depleted serum 2 in terms of anti- ganglioside antibody content using the ganglioside ELISA. Toxic amounts of anti- GM2 antibodies were only detectable in the non-depleted serum 2 (Fig. 9).

Discussion:

This example shows that the present invention is suitable for a therapeutic and/or theranostic approach by identifying body fluids with the capability to kill Ewing sarcoma tumor cells. Furthermore GM2 as the target of these body fluids on Ewing sarcoma cell lines have been identified with the present invention and can be used as a target for further therapeutic and diagnostic approaches against Ewing sarcoma. Human sera cytotoxic against Ewing sarcoma cells can be identified by the present invention. The responsible cytotoxic IgM antibodies and their target GM2 have been identified by the ICIS-assay and the ELISA-assay.

With this invention and its theranostic approach patients suffering from Ewing sarcoma and lacking these cytotoxic components in their own serum can be identified. These Ewing sarcoma patients can be treated with the cytotoxic human donor plasma identified in the CISS-assay.

Claims

Claims

A method for the identification of disease-specific molecules of animal or human cells comprising

(a) identifying body fluids from animals or humans not affected by said disease which are cytotoxic for animal or human cells carrying disease- specific molecules;

(b) incubating fractions of said animal or human cells carrying disease specific molecules with cytotoxic body fluids from animals or humans not affected by said disease identified in step (a) and with said animal or human cells carrying disease specific molecules;

(c) identifying fractions that interfere with the cytotoxicity of said body fluids towards said animal or human cells; and

(d) optionally, further fractionating fractions that have tested positive in step (c) and repeating steps (b) and (c) until a homogenous fraction has been identified, said homogenous fraction representing disease-specific molecules; or

(b') splitting said body fluids identified in step (a) into at least two portions and depleting one of said portions from cytotoxicity-mediating molecules;

(c') subjecting molecules from said animal or human cells carrying disease- specific molecules to separation techniques that allow an unambiguous identification of each molecule;

(d') incubating said separated molecules alternatively with a portion of said body fluid not depleted from said cytotoxicity-mediating molecules and with a portion of said body fluid depleted from said cytotoxicity-mediating molecules; and

(e') identifying molecules that are recognized by said portion of said body fluid not depleted from said cytotoxicity-mediating molecules but not by said portion of said body fluid depleted from said cytotoxicity-mediating molecules.

The method of claim 1 wherein said disease-specific molecules are tumor- specific molecules, or associated with allergy, infectious diseases, viral infections (e.g. HIV infections), cardiovascular diseases, neurodegenerative diseases (e.g. multiple sclerosis, Alzheimers disease), immune- and autoimmune diseases (e.g. rheumatic diseases, psoriasis), chronic fatique syndrom, or transplant rejection.

3. The method of claim 1 or 2 wherein said animal cells are mammalian cells.

4. The method of any one of claims 1 to 3 wherein said body fluids are sera, blood or lymph or fractions thereof.

5. The method of any one of claims 1 to 4 wherein said body fluids are body fluids from humans.

6. The method of any one of claims 2 to 5 wherein said tumor-specific molecules are molecules derived from adenocarcinoma, neuroblastoma, or Ewing's sarcoma.

7. Method of anyone of claims 1 to 6 wherein the molecules subjected to separation techniques are proteins, DNA molecules, RNA molecules, carbohydrates, lipids, glycolipids, steroids, lectins or peptides.

8. The method of any one of claims 1 to 7 wherein the separation techniques are selected from one or two dimensional gel-electrophoresis and overlay high performance thin layer chromatography (HPTLC).

9. The method of any one of claims 1 to 8 wherein the molecules subjected to separation techniques are subsequently immobilized.

10. The method of claim 9 wherein immobilization is effected by Western blotting, FAR Western blotting or TLC overlay techniques.

11. The method of any one of claims 1 to 10 wherein fractions of said animal or human cells carrying disease specific molecules are protein fractions, ganglioside containing fractions, DNA or RNA containing fractions or carbohydrate fractions.

12. The method of any one of claims 1 to 10 wherein said fractions of said animal or human cells carrying disease specific molecules are membrane fractions, nuclear fractions or cytosolic fractions.

13. The method of any one of claims 1 to 12 wherein said fractions obtained in step (c) or step (d) are obtained by chromatographic methods.

14. The method of any one of claims 1 to 13 wherein said homogenous fraction identified in step (d) consists of a single type of molecule.

15. The method of any one of claims 1 to 13 wherein the cytotoxicity-mediating molecules include antibodies.

16. The method of claim 15 wherein said cytotoxicity-mediating molecules further comprise complement or cytotoxic T lymphocytes.

17. The method of any one of claims 1 to 16 wherein said depletion in step (b') is effected by at least one round of incubation of the cytotoxic body fluid with said animal or human cells carrying disease-specific molecules.

18. The method of any one of claims 1 to 17 wherein said disease-specific molecules are proteins, gangliosides, DNA or RNA molecules, carbohydrates, lipids, glycolipids, steroids, lectins or peptides.

19. The method of any one of claims 1 to 18 wherein said disease-specific molecule identified are further characterized by mass spectrometry.

20. The method of any one of claims 1 to 19 wherein said identification of body fluids in step (a) is effected by high-throughput screening.

21. The method of any one of claims 1 to 19 further comprising identifying an agonist, inhibitor or antagonist of the disease-specific molecule identified.

22. The method of claim 21 comprising incubating said disease-specific molecule identified with one, several or a library of small molecules, peptides, aptamers or antibodies and identifying small molecules, peptides, aptamers or antibodies that interfere with or stimulate the biological activity of said disease-specific molecule or that interfere with or stimulate the binding of said disease-specific molecule with its receptor.

23. The method of claim 21 or 22 further comprising the improvement of the pharmacological properties of said inhibitor or antagonist wherein the further following steps are carried out:

(1 ) identification of the binding sites of the agonist, inhibitor or antagonist and the disease-specific molecule by site-directed mutagenesis or chimeric protein studies;

(2) molecular modelling of both the binding site of the agonist, inhibitor or antagonist and the binding site of the disease-specific molecule; and

(3) modification of the agonist, inhibitor or antagonist to improve its binding specificity for the disease-specific molecule.

24. The method of any one of claims 21 to 23 wherein the pharmacological properties of said agonist, inhibitor or antagonist are further improved by peptidomimetics.

25. The method of any one of claims 21 to 24 wherein the pharmacological properties of said agonist, inhibitor or antagonist is further improved by:

(i) modified site of action, spectrum of activity, organ specificity, and/or

(ii) improved potency, and/or

(iii) decreased toxicity (improved therapeutic index), and/or

(iv) decreased side effects, and/or

(v) modified onset of therapeutic action, duration of effect, and/or

(vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by

(i) esterification of carboxyl groups, or

(ii) esterification of hydroxyl groups with carbon acids, or

(iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof.

26. The method of any one of claims 21 to 25 further comprising formulating the agonist, inhibitor or antagonist identified or improved into a pharmaceutical composition or medical product.

27. The method of any one of claims 1 to 20 wherein the disease-specific molecule identified is the GM2 or the MHH-ES1 ganglioside.

28. Use of a human serum or plasma derived from a healthy donor that comprises antibodies mediating cytotoxicity against cells harboring the GM2 or the MHH- ES1 ganglioside in the preparation of a pharmaceutical composition for the treatment of a malignancy.

29. The use of claim 28 wherein said malignancy is Ewing sarcoma.

30. The method of any one of claims 1 to 20 further comprising the step of formulating the disease-specific molecule identified as a vaccine.

31. A method of identifying tumor-specific antigens on cancer cells characterized in that

(a) human sera with cytotoxic characteristics against cancer cells are identified;

(b) protein fractions from lysed cancer cells are incubated with these cytotoxic sera;

(d ) those protein fractions that inhibit the killing of the cancer cells are identified and purified; or (c2) target antigens are identified via western blot analysis; and (d) subsequently a characterization is effected via mass spectrometry and/or via Edman sequencing.

32. The method of claim 31 wherein, the identification of human sera with cytotoxic characteristics is effected by:

(a) incubation of tumor cells with sera of different origin;

(b) subsequent selective staining of the killed cells; and

(c) subsequent determination of the fluorescence.

33. The method of claim 31 or 32 wherein the protein fractions are obtained from the lysed cancer cells by chromatographic separation techniques and a repeated purification is effected by the respective determination of the inhibition until a homogeneous molecule component is obtained.

34. The method of any one of claims 31 to 33 wherein the human sera with cytotoxic characteristics are deleted from the toxic molecules by repeated adsorption to the cancer cells.

35. The method of any one of claims 31 to 34 wherein the human sera with cytotoxic characteristics are separated from the toxic molecules by repeated adsorption to the cancer cells, that the separation of the total proteins of the cancer cells is effected via gel-electrophoresis and that the identification of target-antigens is effected by comparative western blot analysis.

36. Detection and use of body fluids displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

37. Detection and use of fractions of body fluids displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

38. Detection and use of antibodies, antibody fractions or subclasses of antibodies from human blood serum or plasma displaying cytotoxicity against malignant cells for therapy and diagnosis of human malignancies.

39. Detection and use of body fluids or fractions thereof for the identification of specific targets which mediate cytotoxicity on malignant cells.

40. Use of targets detected according to any one of the preceding claims for therapy and diagnosis of human malignancies.

41. Use of body fluids and the FITT™ Technology for predictive assays in therapy.