DE10240118A1 - Characterizing and identifying proteins specific for renal cell carcinoma, useful for diagnosis, prognosis, prevention and treatment, comprises comparing proteome patterns of healthy and diseased tissue - Google Patents

Abstract

Characterizing and/or identifying proteins (I), specific for renal cell carcinoma (RCC) comprises preparing two extracts (E1, E2) containing soluble proteins, with E1 being from RCC, separating the extracts by two-dimensional (2D) gel electrophoresis to produce proteome patterns (P1, P2), each of many proteins, and comparing P1 and P2. Independent claims are also included for the following: (1) proteome (P) of RCC comprising a pattern of individual proteins produced by the new method; (2) RCC-associated proteins (Ia), optionally modified; (3) antibodies (Ab) directed against (Ia); (4) protein chip containing (Ia); (5) nucleic acid chip containing sequences (II) that encode (Ia); (6) method for identifying low molecular weight agents that affect RCC; and (7) synthetic DNA that encodes (Ia).

Description

The present invention relates refer to a method for characterization and / or identification of proteins that are for monitoring, diagnosis and prognosis of patients with primary renal cell carcinoma, for the Selection of patients who are specific to therapy with G250 antibodies address, as well as therapeutic target structures for the treatment this disease can be used. The proteins or polypeptides or these coding nucleic acids can also used to identify pharmacologically active substances or / and even as immunogens for serve to stimulate specific immune responses.

Human renal cell carcinoma poses the most frequent Kidney neoplasia. However, the molecular mechanisms which lead to initiation and progression in renal cell carcinoma, relatively little clarified. investigations show that it is a complex multi-step process, that with the accumulation of different genetic alterations, including amplifications of the EGF receptor and / or mutations or loss of tumor suppressor genes, such as that of the Hippel-Lindau gene, accompanied.

Only the tumor stage caused by the Tumor spread, involvement of regional lymph nodes and that The presence of distant metastases is characterized today widely accepted by pathologists and urologists as an indicator of patient prognosis. So far, little is over the function of molecular regulation and interaction of genetic Alterations in the development of renal cell carcinoma as well as in known of metastasis. For this reason, there are so far Time neither valid molecular markers for diagnosis, prognosis and that monitoring for therapy response can be used. Such Tumor markers are essential and lead in the long term for more efficient treatment of renal cell carcinoma.

Many of the genes / proteins involved in initiation and progression of renal cell carcinoma are involved, are also not known. Identifying such differential expressed genes in renal cell carcinoma provides the basis for identification of markers for the biological phenomena like invasiveness or metastasis. Again, these are of great clinical importance, in particular for the Diagnosis, prognosis and treatment of renal cell carcinoma.

General is renal cell carcinoma a refractory to therapy Tumor and conventional therapies such as surgery, radiation and Chemotherapy, are relatively unsuccessful. The high spontaneous regression rate, the existence of lymphocytic Infiltrates and some objective remissions in clinical trials with various immunotherapeutic agents indicate that renal cell carcinoma an attractive target for represents different immunotherapeutic strategies. One of these Strategies is treating renal cell carcinoma with one monoclonal antibodies (mAb) directed against G250, with G250 at a high percentage on the cell surface is expressed by clear cell renal cell carcinomas (cf. WO 02/062972). Around Patients on therapy with a mAb directed against G250 is to address, to preselect, is the identification of differentially expressed tumor markers, those after antibody treatment either induced or inhibited, essential.

To express differences in expression between normal cells and cancer cells as well as between untreated and To evaluate treated cancer cells, different mRNA, or protein-based methods are used. These include in addition differential display PCR, cDNA microarrays and DNA chip technology serial analysis of gene expression (SAGE). These methods will be successful for the creation of global and quantitative mRNA expression profiles used by cells and tissues. Disadvantages of these analyzes are the lack of detection of post-transcriptional mechanisms, e.g. (i) protein modifications, (ii) half-life of specific proteins or mRNAs, (iii) intracellular Localization and (iv) molecular association of protein products. For this reason it is complementary to the mRNA expression analysis "Proteomics" to see. This technology can be used in addition to quantitative protein expression profiles Moreover information about provide appropriate protein modifications. Also about protein-protein interactions as well as the subcellular Protein localization can with a suitable test execution Information will be obtained. The proteome analysis is based on this technically on the combination of two-dimensional gel electrophoresis (2DE) for the separation and quantification of proteins and analytical Methods such as mass spectrometry and N-terminal protein sequencing, that enable protein identification.

• (a) Bereitstellen eines ersten und eines zweiten Extrakts, umfassend lösliche Proteine, worin zumindest der erste Extrakt aus Nierenzellkarzinomzellen gewonnen wird,
• (b) Auftrennen des ersten und zweiten Extrakts mittels zweidimensionaler Gelelektrophorese, wobei ein erstes und zweites Proteommuster, jeweils bestehend aus einer Vielzahl von Proteinspezies erhalten wird,
• (c) Vergleichen des ersten und zweiten Proteommusters und
• (d) Charakterisierung oder/und Identifizierung von Nierenzellkarzinom spezifischen Proteinen.

An object of the present invention was to provide tumor markers for the identification, monitoring and treatment of renal cell carcinomas. This object is achieved according to the invention by a method for characterizing and / or identifying proteins specific to renal cell carcinoma, which comprises the following steps:

• (a) providing a first and a second extract comprising soluble proteins, wherein at least the first extract is obtained from renal cell carcinoma cells,
• (b) separating the first and second extract by means of two-dimensional gel electrophoresis, a first and second proteome pattern, each consisting of a large number of protein species, being obtained,
• (c) comparing the first and second protein patterns and
• (d) Characterization and / or identification of renal cell carcinoma specific proteins.

An object of the present invention is a method which is suitable by means of a differential Proteome analysis to identify renal cell carcinoma specific tumor markers the for an optimized diagnosis, prognosis and / or treatment of renal cell carcinoma patients can be used.

Proteins are shown in Table 2, those in renal cell carcinoma cells compared to healthy kidney cells are expressed differentially. One or more of these proteins, in particular at least 5, more preferably at least 10 of these genes, can used to diagnose the condition of kidney cells. The Proteins shown in Table 2 are particularly suitable as molecular markers for the diagnosis or prognosis of renal cell carcinoma. In particular are the genes shown in Table 2 upon initiation or / and Progression of renal cell carcinoma involved. So you can as a marker for biological phenomena, such as invasiveness or metastasis, are used and are therefore of great importance for the Diagnosis, prognosis and treatment of renal cell carcinoma.

The proteome analysis used according to the invention is based in particular on the combination of two-dimensional gel electrophoresis for the Separation and quantification of proteins, coupled with analytical Methods for protein identification, such as mass spectrometry and protein sequencing.

The proteins found in the process, which are shown in Table 2 can be used in particular as tumor markers or used as a therapeutic target structure. Furthermore can these tumor markers in immunoassays, like in protein chip manufacture, Find use. additionally can also the coding nucleic acids for DNA chip production Find use.

The identified tumor markers fall essentially in the following subgroups: involved in metabolism Proteins, proteins associated with proliferation / apoptosis, structure / cytoskeletal proteins, Oncogene / tumor suppressor gene associated proteins, transporters of Substances, adhesion proteins, Protein processing / degradation proteins, tumor antigens, stress proteins, putative tumor antigens and unknown proteins.

An object of the present invention is the identification of new tumor markers. At least one of the proteins summarized in Tables 1, 2 and 3, used as a general tumor marker or therapeutic target structure become. The tumor markers listed in the table should be specific be used in renal cell carcinoma. One more way consists in using these tumor markers in immunoassays as well in protein and DNA chip production, which is then used again to monitor a large patient base can be.

Another object of the invention is the identification of tumor markers in renal cell carcinoma, their expression by treatment with antibodies directed against G250 are influenced. Frequency analysis is a prerequisite of G250 surface expression on renal cell carcinoma cell lines and normal renal epithelial cells Flow cytometric analysis of 35 renal cell carcinoma cell lines revealed that approximately 50% of the clear cell renal cell carcinoma cell lines analyzed, but not autologous normal renal epithelia G250 antigen on the cell surface expressed. G250 expression varied with a specific one medium fluorescence intensity (MFI) of 2.6-33.2, however, strongly between the individual renal cell carcinoma cell lines. This resulted for the classification of "Iow", "medium" and "high" -G250 "expressors".

In the next step, corresponding "low", "medium" and "high" G250 expressing cell lines for treated at different times with antibodies against G250 and the protein expression pattern of untreated with that of antibodies compared to G250-treated tumor cells. The differential expressed proteins were identified using 2DE and mass spectrometry were used as methods. The identified proteins are summarized in Table 1 and can be used as tumor markers in immunoassays also for protein and DNA chip production, the use of which is then a preselection of a big one Patient base regarding enables a putative response to antibody-based therapy, be used.

When using the method according to the invention identified and characterized proteins are in the essential around

• - structural proteins
• - chaperones
• - signal transduction proteins
• - at the Proteins involved in metabolism
• - at the Protein degradation / processing involved proteins
• - on Proteins involved in apoptosis / cell cycle
• - transporter proteins
• - tumor antigens
• - immunophilins
• - unknown Proteins.

Here, at least 1.5 times regulated, in both positive and negative directions, statistically significant differentially regulated proteins are taken into account.

The proteins shown in Table 1 can in particular. to monitor the success of a treatment with antibodies against G250. In addition, they can to be used in combination products for the treatment of renal cell carcinoma provide. Such combination preparations contain in addition to the antibody against G250 prefers another active ingredient, which is the major vault protein, a protein selected from Table 3a or 3b, or selected a protein from Vimentin, Moesin, Heat shock 27 kD Protein 1, Heat shock 90 kD Protein 1 beta or aldehyde reductase influenced. Through the targeted Combination, based on the found, differentially expressed Proteins, on the one hand, can increase the success of treatment, on the other hand if necessary, the formation of undesirable side effects can also be suppressed.

To further clarify the present The following examples, figures and tables are used in the invention.

1 shows G250 expression in different renal cell carcinoma cell lines.

2 shows FACS diagrams of two different G250 phenotypes in renal cell carcinoma.

Table 1 shows proteins that are found in with an antibody G250 treated renal cell carcinoma cell lines differentially from untreated Renal cell carcinoma cell lines are expressed.

Table 2 shows in renal cell carcinoma lesions Proteins differentially expressed compared to healthy renal epithelium.

Table 3 shows significant subgroups of G250 regulated proteins.

Example 1:

Expression of G250 the surface of renal cell carcinoma cell lines to select suitable cell lines for Detection of G250-regulated tumor markers (Table 1, 3)

(i) FACS analysis

A total of 35 human renal cell carcinoma cell lines derived from surgically removed renal tumor tissue and autologous renal cell epithelia were analyzed for G250 surface expression. In 18 of 35 renal carcinoma cell lines, G250 surface expression was detected by flow cytometry, the expression of which varied greatly between the different cell lines ( 1 and 2 ).

For the following proteome analyzes were so far 4 different G250 expressing Renal cell carcinoma cell lines selected: one "low", one "medium" and two "high" expressors. These cells were grown in the presence or absence of G250 (5 μg / ml) for 24 or Incubated 48 hours and 21 days before protein extracts were made were.

(ii) 24 hour treatment of the renal cell carcinoma cell lines with G250

• - cells plate out so that approx. 50% confluent
• - 1 Leave day in medium
• - encore of new medium, including 5 T125 bottles untreated, 5 T125 bottles with G250 5.3 μg / ml (corresponds to 30 μ1 [5 mg / ml] on 28 ml medium)
• - to 24 hour harvest of cells
• - 2 * wash in PBS
• - Trypsinize
• - 3 * wash in PBS
• - Got over lose weight
• - dry Pellet in liquid Nitrogen, then at -80 ° C to store

(iii) 21 day treatment of the renal cell carcinoma cell lines with G250

Cells are not split, medium change according to the following plan
Day 1: cell seeding, medium + G250
Day 4: addition of G250
Day 8: Change of medium + G250
Day 11: addition of G250
Day 14: Add G250
Day 15: Change of medium + G250
Day 19: addition of G250
Day 22: addition of G250
Day 23: harvest
G250: Add 30 μl of a 5 mg / ml solution to 25 ml medium in a T175 bottle

Cells are split
Day 1: 5 x 106 cells / T125 bottle ± G250
Day 4: addition of G250
Day 8: Medium change + G250 / split
Day 11: addition of G250
Day 14: Add G250
Day 15: Change of medium + G250 / split
Day 19: addition of G250
Day 22: Add G250 / split
Day 23: harvest
G250: Add 30 μl of a 5 mg / ml solution to 25 ml medium in a T175 bottle

sample material

Treated samples are used or untreated renal carcinoma cell lines, e.g. MZ 1846RC. For statistical As a safeguard, five 2D gel replicates are made from each sample, which is then computer densitometric be evaluated comparably.

sample preparation

The cell pellet is in liquid nitrogen with a mortar finely ground. 3 mg are dissolved in 200 μL lysis buffer. With the suspension is treated on the ultrasonic rod while cooling in an ice water bath (10 × 0.5 s). Now with 800 μl Diluted lysis buffer and shaken at room temperature for 30 minutes. Finally, 10 Minutes at 20 ° C and 14000 xg centrifuged, the supernatant removed and portioned frozen at -80 ° C. The protein concentration is determined using the Bradford method.

Example 2 (Table 2):

Tumor marker of renal cell carcinoma, which are differential in renal cell carcinoma cells and normal kidney cells be expressed

sample material

It will be kidney tissue from six patients compared, three of which were kidney tumor negative and three kidney tumor positive were. For the statistical validation, five proteome exhibits are made from each sample, which then be comparatively evaluated by computer densitometry.

sample preparation

The kidney tissue is in liquid nitrogen with a mortar finely ground. 3 mg are dissolved in 200 μL lysis buffer. With the suspension is treated on the ultrasonic rod while cooling in an ice water bath (10 × 0.5 s). Now with 800 μl Diluted lysis buffer 1 and shaken at room temperature for 30 minutes. Finally, 10 Minutes at 20 ° C and 14000 xg centrifuged, the supernatant removed and portioned frozen at -80 ° C. The protein concentration is determined using the Bradford method.

Example 3

proteome

Rehydration of the focus strips (Immobiline Dry Strips)

The focus strips (T = 4 %, C = 3%, pH 4-7) are delivered in the dried and frozen state. For use they are about Swollen in rehydration buffer overnight in a reswelling tray. For one 18 cm long strips are used with 400 μL rehydration buffer.

Focus (first dimension)

The cooling of the focusing apparatus is set to 20 ° C. After swelling, the strips rehydrated overnight are briefly immersed in double-distilled water and placed on a damp electrode paper, which in turn lies on a dry filter paper. Now place another damp filter paper and a dry filter paper on the strip. Now apply light pressure to remove the excess moisture from the strip. After placing the focusing strips in the focusing chamber, the sample is applied to the gel. The sample solutions are colored with bromophenol blue to check the tightness of the application cups. The focus runs overnight and is finished after 38.7 kVh. Focus program for pH 4–7:

Pour the gels for the second dimension

The 1.5 l gel solution for gel pouring is the day before manufactured and degassed. The is in a sealed bottle Overnight solution in a refrigerator to approx. 4 ° C cooled. The reservoir of the casting chamber is closed with a funnel and a glycerin solution in it (500 ml). The prepared gel solution 3 is removed from the refrigerator directly before the casting taken and 72 ul TEMED or 7.2 ml APS solution stirred and this solution filled into the casting chamber through a funnel. The entire gel solution is in the chamber, the funnel is removed and the glycerin solution is left out enter the reservoir. This expresses the gel solution the filling hose and the lower part of the casting chamber, so that the gel solution only is still in the cast cassettes. Then be as quick as possible the gels with water-saturated Layered butanol 7. The amount of gel solution and the glycerin solution 6 depends on the number of cast cassettes used and is also dependent the volume of the casting chamber. After three hours you can polymerize Remove the gels from the cassette.

Second dimension

In the Hoefer Dalt chamber the Tank buffer (20 l) mixed and to about 13 ° C cooled. Until use, the gels in their cassettes are double-distilled Water layered. On top of each gel is a small strip of filter paper with 5 μL a protein standard solution added (Marker). The frozen focus strips are first in a 10 ml test tube (for two strips in a test tube) of a fresh DTT solution 10 Minutes equilibrated. After that, the solution exchanged for 10 ml of a fresh bromophenol blue-colored iodoacetamide solution and shaken again for 10 minutes.

The water in the cast cassettes will poured, the focused IPG strips briefly in the tank buffer dipped and applied horizontally to the gel.

Markers and IPG strips are now overlaid with a hot agarose solution. As soon as the agarose has set, the gels can be installed in their cassettes in the Hoefer Dalt chamber. Excess tank buffer is removed and the electrophoresis program started. The running-in phase (step 1) runs 50 Vh. The main phase (step 2) continues until the bromophenol blue front has reached the lower end of the gel. The gels are now removed from the cassettes and drawn through double-distilled water. Elektrophoreseprogramm:

Staining of the gels

Each gel is in its own bowl colored. First it is fixed with 330 ml of an ethanol / acetic acid mixture for 30 minutes. The SyproRuby solution (330 μl) is then stained for three hours with the light closed. With 330 ml of the ethanol / acetic acid mixture the background is then decolorized for 30 minutes with the light closed. In front the gel is scanned twice after scanning with the fluorescence scanner double-distilled water is drawn to remove the last residues of the dye remove. After scanning, the gel is placed in a colloidal Coomassie staining solution overnight shaken. The next Day will be colored Gels shaken in double-distilled water. Is the background decolorized accordingly, the gel can be scanned with the visual scanner.

Computer densitometry of the 2D gels

• – Standardabweichung ein- und desselben Spots innerhalb einer Gruppe ist ≤ 30 %
• – parametrischer Signifikanztest (student T-Test) wird angewendet
• – die Spotveränderung zwischen 2 Gruppen weist einen „confidence level" von ≤ 0,05 auf
• – Regulationsfaktor eines Spots zwischen 2 Gruppen ist ≥ 1,5

The gels are evaluated using the PD Quest image analysis software (version 6.2.1, Bio Rad). For this, 5 2D gels per group are matched against each other. The resolution of the Tiff files of the gels (16bit) is 254 dpi. Such spots are considered significant if they meet the following criteria:

• - Standard deviation of one and the same spot within a group is ≤ 30%
• - Parametric significance test (student t-test) is used
• - The spot change between two groups has a "confidence level" of ≤ 0.05
• - The regulation factor of a spot between 2 groups is ≥ 1.5

In addition, such spots deemed significant that a complete deregulation between Have 2 groups. Thereby within the group of the existing Spots also standard deviations> 30 % authorized.

MALDI - TOF mass spectrometry (MS) of the target proteins

The detected by computer densitometry Target proteins are now cut out of the polyacrylamide gel and digested with trypsin. The peptides obtained are treated with acetonitrile / 0.1 % TFA eluted from the gel and then desalted with a C18 ZipTip column. These extracts are now cocrystallized with 2,5-dihydroxybenzoic acid and pipetted onto a MALDI-MS target plate.

These crystallized peptides will subsequently in the MALDI-TOF mass spectrometer (Voyager STR, Applied Biosystems) ionized with an N2 laser (337 nm). The ions are caused by voltages from 20-25 kV accelerates and at the same time their flight time, which is mass-specific is measured. After evaluating the resulting spectra, leave identify the individual proteins through database searches.

Claims (45)

A method for the characterization and / or identification of renal cell carcinoma-specific proteins, comprising the following steps: (a) providing a first and a second extract comprising soluble proteins, wherein at least the first extract is obtained from renal cell carcinoma cells, (b) separating the first and second Extracts by means of two-dimensional gel electrophoresis, whereby first and second proteome patterns, each consisting of a multiplicity of protein species, are obtained, (c) comparing the first and second proteome patterns and (d) Characterization and / or identification of renal cell carcinoma specific proteins. A method according to claim 1, characterized in that the first extract from renal cell carcinoma cells, preferably from tissue biopsies from patients, and the second extract obtained from normal kidney cells becomes. A method according to claim 1, characterized in that the first extract from untreated and the second extract from treated Renal cell carcinoma cells are obtained. A method according to claim 3, characterized in that for Treatment of renal cell carcinoma cells polyclonal and / or monoclonal Antibody, Antibody fragments, single-chain, bispecific, chimerized or / and humanized antibody used at least against a renal cell carcinoma specific Antigen. A method according to claim 4, characterized in that the specific antigen is G250. Method according to one of claims 3 to 5, characterized in that that the antibodies used to treat the antigen G250 are directed. Method according to one of claims 3 to 6, characterized in that that the antibody the chimeric monoclonal antibody cG250 is. A method according to any one of claims 1 to 7, wherein the two-dimensional Gel electrophoresis includes the following steps: (a) Separation of the extracts in a first dimension corresponding to the isoelectric point (b) followed by a split into a second Dimension according to the size. A method according to any one of claims 1 to 8, wherein renal cell carcinoma specific protein species are selected which (a) on different positions on the two-dimensional gels, from the first and second extract are obtained, are localized or and (b) different intensities on the two-dimensional Have gels of the first and second extract. Method according to one of claims 1 to 9, characterized in that that the protein species are characterized by peptide fingerprinting become. Method according to one of claims 1 to 9, characterized in that that the protein species are characterized by mass spectroscopy are or / and are at least partially sequenced. Method according to one of claims 1 to 1 1, characterized in that that the cells from which the extracts are obtained are mammalian cells are. A method according to claim 12, characterized in that the cells used are human cells. A method according to any one of claims 1 to 13, wherein the renal cell carcinoma specific protein species are selected from serum proteins, Structural proteins, signal transmission proteins, Proteasome subunits, stress factors, tumor antigens, oncogenes, Proteins associated with apoptosis and proliferation, transporter proteins, adhesion proteins or / and proteins associated with metabolism and protein processing. Method according to one of claims 1 to 14, which the additional Step includes: (e) characterization and / or identification of kidney cell carcinoma-specific proteins in a kidney cell carcinoma sick patient. Proteome of a renal cell carcinoma cell, consisting of a pattern of individual proteins, which are processed using a method according to a of claims 1 to 15 available is. A proteome according to claim 16 containing at least a portion of the proteins shown in Table 1, 2 or 3. Renal cell carcinoma-associated proteins or / and modified Proteins selected from the group of serum proteins, structural proteins, signal transmission proteins, Proteasome subunits Stress factors, tumor antigens, oncogenes, apoptosis and profliferations associated proteins, transporter proteins, adhesion proteins and / or metabolism and protein processing-associated proteins. Renal cell carcinoma-associated proteins or / and modified Proteins as indicated in Table 1, 2 or 3 or proteolytic Fragments of it. Antibody, directed against a protein according to one of claims 18 or 19. Use of a proteome according to claim 16 or 17 or / and of a protein according to one of claims 18 or 19 or / and one antibody according to claim 20 for the diagnosis, prognosis, prevention or therapy for renal cell carcinoma. Use of a proteome of claims 1'6 or 17 or / and of a protein according to one of claims 18 or 19 or / and one antibody according to claim 20 for the identification of pharmacologically active substances. Use of the coding nucleic acid sequence or / and a variant thereof a protein according to one of claims 18 or 19 for diagnosis, Prognosis, prevention, Therapy for renal cell carcinoma and / or the identification of pharmacologically active substances. Use according to claim 23, characterized in that the nucleic acid a DNA or RNA, preferably a DNA, in particular a double-stranded DNA, is. Use according to claim 23, characterized in that the nucleic acid has its antisense sequence. Use of at least one of the proteins according to one of the Expectations 18 or 19 or / and at least one antibody according to claim 20 for Production of a protein chip assay for diagnosis and / or prognosis of renal cell carcinoma, especially for monitoring patients. Protein chip, available according to claim 26. Use of at least one coding nucleic acid sequence or / and a variant thereof of a protein according to one of claims 18 or 19 for the production of a nucleic acid chip assay, in particular a DNA chip assay, for the Diagnosis and / or prognosis of renal cell carcinoma, especially for Patient monitors. Nucleic acid chip, available according to claim 28. Use of antibodies against one of the proteins from one of claims 18 or 19 for production an immunoassay for the diagnosis or / and prognosis of renal cell carcinoma, in particular for monitoring patients. Use of antibodies against at least one of the proteins from one of claims 18 or 19 for the manufacture of a pharmaceutical composition for treatment of renal cell carcinoma. Use of polyclonal antibodies, monoclonal antibodies, F (ab ') ₂ , Fab' or Fab antibody fragments, single-chain antibodies, bispecific antibodies, chimerized antibodies or / and partially or completely humanized antibodies against one of the proteins according to one of claims 18 or 19 for the production of a pharmaceutical composition in combination with cytokines, selected from the group consisting of: interleukins IL-2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, and 15, Interferons IFN-α, IFN-β and IFN-γ, tumor necrosis factor TNF-α, TNF-β, nerve growth factor (NGF), ligands of CD40, FAS, CD 27 and CD 30, macro-inhibiting protein (MIP), Rantes, active fragments thereof and / or pharmaceutically suitable analogs and derivatives as well as suitable combinations for the treatment of patients suffering from renal cell carcinoma. Use of antibodies, monoclonal antibodies, F (ab ') ₂ , Fab' or Fab antibody fragments, single-chain antibodies, bispecific antibodies, chimerized antibodies and / or partially or completely humanized antibodies against one of the proteins according to one of claims 18 or 19 for the manufacture of a pharmaceutical composition in combination with low molecular weight active substances (molecular weight ≤ 2000 Da) for the treatment of renal cell carcinoma sick patient. Use of antibodies, monoclonal antibodies, F (ab ') ₂ , Fab' or Fab antibody fragments, single-chain antibodies, bispecific antibodies, chimerized antibodies or / and partially or completely humanized antibodies against one of the proteins from one of claims 18 or 19, characterized in that they are in the form of conjugates with a labeling group and / or cytotoxic group. Use according to claim 34, characterized in that the cytotoxic group is selected from radionuclides and toxins. Pharmaceutical composition, characterized in that it comprises as active component: (a) an antibody, monoclonal antibody, F (ab ') ₂ , Fab' or Fab antibody fragment, single-chain (single chain) antibody, bispecific antibody, chimerized antibody or / and partially or completely humanized antibody which is directed against the antigen G250 and (b) additionally an active ingredient, in particular another antibody or / and a low molecular weight active ingredient which influences at least one protein from Table 1 in such a way that the pharmaceutical composition influences the therapeutic Effect of a first antibody or derivative thereof alone, positively influenced (enhanced). Pharmaceutical composition according to claim 35 to 36, characterized characterized that the antibody used is cG250. Pharmaceutical composition according to one of claims 36 or 37, characterized in that the further active ingredient is the major vault protein (MVP) affects. Pharmaceutical composition according to one of claims 36 or 37, characterized in that the further active ingredient proteins, selected from Table 3a or 3b. Pharmaceutical composition according to one of claims 36 to 39, characterized in that the further active ingredient at least one of the major vault protein (MVP, vimentin (NCBI 3402191, moesin (NCBI 4505257), Heat shock 27 kD Protein 1 (NCBI 4504517), Heat shock 90kD Protein 1 beta (NCBI 6680307) or aldehyde reductase (NCBI 1633300) influenced. Process for finding low molecular weight active substances, which Renal cell carcinoma cells, characterized in that a proteome is used to determine the low molecular weight active substances Claim 16 or 17 or / and a protein according to one of Claims 18 or 19 or / and an antibody used according to claim 20. Synthetic DNA coding for a protein according to one of the Expectations 18 or 19. Protein according to claim 18 or 19, characterized in that it is a recombinantly produced protein. Use according to a protein according to claim 18 or 19 or of a recombinant peptide according to claim 43 for the production of antibodies directed against these proteins. Use of an antibody according to claim 44 for the preparation of a diagnostic or / and therapeutic For the treatment of renal cell carcinoma.