WO2015155217A1

WO2015155217A1 - Method for the purification of natural cytotoxic igm antibodies

Info

Publication number: WO2015155217A1
Application number: PCT/EP2015/057586
Authority: WO
Inventors: Frank PLÖGER
Original assignee: Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh
Priority date: 2014-04-09
Filing date: 2015-04-08
Publication date: 2015-10-15
Also published as: DE102014206881A1

Abstract

The invention relates to a preparation of cytotoxic IgM antibodies which is essentially free from non-cytotoxic IgM antibodies and other classes of antibodies, to a method for preparing such a preparation of cytotoxic IgM antibodies and to a pharmaceutical composition containing a cytotoxic IgM antibody preparation according to the invention, which is advantageously used for the treatment of cancers.

Description

Process for the purification of natural cytotoxic IgM antibodies

DESCRIPTION

The present invention relates to a preparation of cytotoxic IgM antibodies, which is substantially free of non-cytotoxic IgM antibodies and antibodies of other classes, a method for producing such a preparation of cytotoxic IgM antibodies and a pharmaceutical composition comprising a preparation according to the invention of cytotoxic IgM antibodies, which is preferably used for the treatment of cancer.

The fact that human serum has cytotoxic components against a number of tumor cells has long been known (Bolande, 1960). The rate at which cell death is induced by such components suggests complement-mediated cell lysis in conjunction with IgMs. So-called natural IgM antibodies to neuroblastoma (NB) were found in healthy blood donors, but not in patients suffering from neuroblastoma (Erttmann et al., 1996, Ollert et al., 1996). The use of anti-NB IgM containing sera or purified antibodies in nude rats resulted in tumor growth arrest following inoculation of human neuroblastoma cells (David et al., 1996, Ollert et al., 1997). Schmitt et al. (1999), preliminary data from 6 neuroblastoma patients reported from a clinical phase I / II study that serum toxicity increased significantly after plasmapheresis of cytotoxic anti-NB antibodies and tumor necrosis was observed in 2 patients. The use of cytotoxic IgM antibodies is also described in other tumor types. In a melanoma patient, injection of cytotoxic plasma into metastases led to rapid tumor necrosis, which was attributed to massive local complement activation (Schwartz-Albiez, 2007, Schwartz-Albiez et al., 2008). In addition to serums from healthy donors, recombinant monoclonal IgM antibodies have also been developed and used clinically. Such an antibody was used in a clinical study in 50 gastric carcinoma patients Tried and led in 80% of cases to apoptosis of tumor cells within the tumor tissue.

Using a specially developed cell-based screening method, it was shown that, depending on the type of tumor, about 0.25 to 5% of the donor plasmas contained specific cytotoxic IgM antibodies to various tumors (Schwartz-Albiez, 2007).

The previous strategy for producing a clinically useful preparation is based on the pooling of individually identified cytotoxic plasmas. An essential limitation of this method is the cost-intensive screening method, which can not be provided with sufficient material for longer-term therapy of larger groups of patients especially in tumor types against which only low frequency cytotoxic sera are found.

However, such cytotoxic IgM antibodies are found in the same prevalence as in the population in the pooled plasma fractions of all blood product manufacturers, without being able to be used so far.

The object of the present invention was therefore to provide on the one hand by selective enrichment of cytotoxic antibodies from the unused secondary fractions of conventional blood plasma fractionation, a large pool of tumor-specific antibodies available, which can be used for the development of tumor therapeutics. In particular, the object was to provide isolation methods which allow to specifically use the cytotoxic IgMs from unused blood plasma fractions and thus to enable an economically efficient development of the raw material pool of unused blood plasma fractions. On the other hand, the object was to provide pharmaceutical compositions that allow a fundamental treatment of patients with tumors of various forms. The object of the invention is achieved by providing preparations of cytotoxic IgM antibodies, which are substantially free of non-cytotoxic IgM antibodies and of antibodies of other classes.

Substantially free of non-cytotoxic IgM antibodies and antibodies of other classes in the context of the present invention means that the cytotoxic IgM antibodies are present in a purity of more than 80% and other blood plasma constituents are present at not more than 20% in the preparation.

In preferred embodiments, the purity of the cytotoxic IgM antibodies in the preparation according to the invention is more than 85% and more preferably more than 90% as defined above. The purity is determined in the context of the present invention, in particular by HPLC analysis method.

In particular, as stated above, the object of the present invention is to provide a novel purification method which permits the isolation of IgM natural and human immunoglobulins, wherein cytotoxic IgM antibodies are isolated and concentrated to be particularly effective in the treatment of cancers suitable. The cytotoxic effect of the IgM preparations according to the invention can be detected in cell culture models with human cancer cells.

In the prior art, the targeted separation and purification of the natural cytotoxic IgM fraction is not described. Rather, the IgM fraction has hitherto usually been isolated in its entirety and used predominantly as an immune stimulant. For cancer research, on the other hand, mainly recombinant monoclonal cytotoxic IgMs were used. It is also known that out of about 100 individually tested plasmas, more than 90% have no natural cytotoxicity in the cell culture assay. Furthermore, so far only methods for the purification of the complete IgM fraction are known, so no method for the special purification of cytotoxic IgM fractions. The isolation of normal IgM fractions is carried out as described above by the method of Pool, 1965 and Cohn, 1940 and based on the classical plasma fractionation, in which after performing certain steps, an IgM-containing fraction is obtained, which, however, only to a very limited extent cytotoxic Contains IgM antibody.

FIG. 1 shows a scheme of the classical plasma fractionation of the known methods according to Pool and Cohn. In contrast, preparations of cytotoxic IgM antibodies according to the invention are isolated by a combination of chromatographic methods and screening methods. A further subject of the present application are therefore cytotoxic IgM antibody preparations which are obtainable by isolation from blood, serum or plasma from donors, in particular healthy donors, in which the liquid blood components are suitably subjected to a first chromatographic separation,

Chromatography fractions are examined for their cytotoxicity in a cell culture assay with tumor cells, cytotoxic fractions optionally subjected to further purification and separation steps and by rechromatographic separation a fraction is isolated, which contains more than 80% of existing blood plasma components cytotoxic IgM antibody.

The procedure or method by which the cytotoxic IgM antibody preparation of the present invention is available selects similar steps as compared to prior art methods, but it is already aimed essentially at isolation of a cytotoxic fraction in its first step just to optimize the cytotoxic IgM antibody. In addition to further purification steps, the further cleavage of this fraction carried out in a further chromatographic, in particular size-exclusion, chromatographic step makes it possible to concentrate and isolate only the cytotoxic IgM antibodies to a high degree. Thus, while in principle a cytotoxic total fraction is obtained in the conventional purification of blood plasma, in which also contain cytotoxic IgMs, they are in impure and not concentrated form and these fractions are discarded in the usual plasma fractionation. The procedure according to the invention makes it possible to obtain both the largest possible cytotoxic fraction and from this then the enrichment of the cytotoxic IgM antibodies in particular. Without accompanying screening via a cell culture assay, in which the cytotoxicity of the fractions obtained after the first chromatographic treatment is examined, sufficient purification and accumulation of cytotoxic IgM antibodies is not possible.

In the context of the present invention, it is preferred to carry out ion exchange chromatography with anion exchange materials as the first chromatographic treatment, the use of diethylaminoethylcellulose as chromatography material being particularly preferred. Diethylaminoethylcellulose (DEAE-C) is a frequently used, weakly basic anion exchanger, which can be used in particular for the chromatographic separation and purification of high molecular weight proteins and is also used in classical plasma fractionation for the first chromatography step. By means of the first chromatographic purification step, a rough separation of the blood plasma into fractions has taken place. FIG. 2 shows a DEAE chromatogram and the fractions obtained therefrom, which are designated by the numbers 1 to 22.

Such chromatographic fractions are then assayed using a cytotoxicity assay. For this purpose, the effect of the isolated fraction on one or more cancer cell lines is examined. FIG. 3 shows by way of example the performance of a corresponding test with the neuroblastoma cancer cell line Kelly. Kelly cells are publicly available, e.g. from Sigma-Aldrich®, No. 921 1041 1.

For this purpose, a propidium iodide (PI) assay is preferably carried out with the selected plasmas and compared to the control or negative plasmas or negative antibodies used to assess the mean cytotoxicity. Fractions with significantly increased control over average cytotoxicities, preferably with mean cytotoxicity of at least 30 and more preferably at least 40% in the Pl assay are used for further purification.

According to the invention, further purification steps can follow the first chromatographic separation and after selection of the fractions to be used further, such as ultrafiltration and / or ethanol precipitations. In the further chromatography step carried out according to the invention, in particular in the further size exclusion chromatography step, the cytotoxic IgM antibodies are then separated again from other plasma constituents and in particular from non-cytotoxic IgM antibodies. FIG. 4 shows a representation of SEC chromatography and the peak of the active, cytotoxic IgM antibody moiety. The isolation of the corresponding fractions assigned to this peak leads to a highly purified and highly concentrated preparation of cytotoxic IgM antibodies. Preferably, only such a fraction of the fractions is further used after the chromatography that the desired cytotoxic IgM antibodies to more than 80, preferably more than 85 and more preferably more than 90%, based on other plasma protein portions, are present in these fractions.

The preparation of cytotoxic IgM antibodies thus obtained is particularly well suited for pharmaceutical purposes. For the purification method according to the invention, the use of blood plasma or serum of a single donor is just as possible as the use of whole plasma pools. Especially with individual donors whose blood is known to have high concentrations of cytotoxic IgM antibodies, the purification makes sense. However, only 0.25 to 5% of healthy blood plasma donors have specific cytotoxic IgM antibodies to various tumors (Schwartz-Albiez, 2007). In this respect, the screening of individual donors for the presence of specific cytotoxic IgM antibodies extremely expensive and does not give a high positive rate. Therefore, it is preferable to resort to the existing blood plasma pools, due to the heterogeneous donor composition and a corresponding proportion of donor blood or donor plasmas with cytotoxic IgM antibodies is to be expected. The purification process according to the invention makes it possible to safely isolate those fractions which have IgM antibodies and to concentrate and purify the antibodies from high volumes of blood, plasma or serum.

The cytotoxic IgM antibody preparations according to the invention are very particularly preferably obtainable by an optimized process in which a DEAE chromatography at a pH of 7.8 to 8.8, preferably 8 to 8, from a blood product, in particular plasma, after concentration and appropriate buffering 8.5 and more preferably 8.2 to 8.3 is performed. The resulting fractions containing cytotoxic IgM antibodies are determined by the aforementioned cytotoxicity test with cancer cell lines, such chromatographic fractions with suitable cytotoxicity values, which indicate the presence of cytotoxic IgM antibodies, subjected to further purification steps. Preferably, an ultrafiltration is first carried out according to standard methods, in which a suitable size exclusion is set, which allows a purification of IgM antibodies, which allow due to their pentameric or hexameric structure and their molecular weight of about 900 kD.

In preferred embodiments, ultrafiltration is followed by ethanol precipitation. This ethanol precipitation is carried out in aqueous, 7 to 1 1% ethanolic solution and preferably 9 to 10% ethanol solution with pH of 4.5 to 6.5 and preferably 4.8 to 5.8, particularly preferably 5.1 to 5, 3 performed.

This is followed in a particularly preferred embodiment, a renewed ultrafiltration of the supernatant of ethanol precipitation, for which the same conditions as apply to the above-mentioned first ultrafiltration in terms of their size exclusion. With a particularly preferred second ethanol precipitation, renewed ethanol precipitation, now in 20 to 30% strength ethanolic solution, preferably 22 to 27% and particularly preferably 23 to 25% strength solution, at a higher ethanol concentration, is preferred at a pH of 5.9 to 8 , 5, more preferably 7 to 7.5, and most preferably 7.2 to 7.3.

After centrifugation and subsequent dissolution of the sediments in an aqueous buffer solution, preferably a sodium acetate solution, at a pH of preferably 5 to 6.5, more preferably 5.5 to 6.2 and most preferably 5.8 to 5.9 become the so Samples subjected to size exclusion chromatography (SEC chromatography). In this case, those fractions which can be assigned to the cytotoxic IgM-containing part of the blood plasma fraction are isolated and, where appropriate, reconstituted by suitable methods, e.g. Ultrafiltration, concentrated to the corresponding end product. Size exclusion chromatography may be carried out with suitable buffers which, in terms of both their contents and their pH, allow eventual use of the concentrated selected IgM fractions for direct administration to a patient.

The preparations according to the invention can thus be easily prepared by standard methods from sources available in large quantities. Due to the relatively high levels of cytotoxic IgM antibodies contained in them, effective treatment of patients, especially tumor patients, is possible with the aid of these preparations. The fact that the cytotoxic IgM antibody preparations according to the invention have high purity and contain only very small amounts of other blood or plasma constituents also makes these preparations a very safe means of treating patients. Apart from the particularly preferred method of obtaining the preparations of cytotoxic IgM antibodies from donor blood, plasma or serum from individuals or from the plasma pools of blood donation services, there is also the possibility according to the invention of donors with known high concentrations of cytotoxic IgM antibodies B To isolate cells and to cultivate these B cells and to isolate them specifically cytotoxic IgM antibodies.

Compared to monoclonal antibodies of the IgM type, the cytotoxic IgM antibodies of the present invention have the advantage that they are polyclonal, so that they can be used against a higher number of tumor diseases. Due to the polyclonality they are also multispecific.

Another object of the present invention is a process for preparing a preparation of cytotoxic IgM antibodies, as described above in the context of the corresponding preparations. Of course, preferred embodiments described therein also apply to the method itself.

Another object of the present invention are pharmaceutical compositions containing preparations of cytotoxic IgM antibodies of the type described herein. Preferably, these cytotoxic IgM antibodies are formulated together with pharmaceutically acceptable excipients and additives. As already described above, such pharmaceutical compositions are particularly suitable for use in the treatment of tumor diseases. Cytotoxic IgM antibodies bind in particular to cell surface structures, as they have tumor cells, but also bacteria. Glycosylation of these cells is believed to cause binding of the antibodies thereto, whereas normal or low binding usually does not occur to normal cells. In combination with complement, the cytotoxic IgM antibodies cause destruction of the tumor cells. The cytotoxic IgM administered according to the invention with the pharmaceutical composition Antibodies interact with the complement present in the body, whereby the walls of the tumor cells are perforated, which ultimately leads to lysis of the cells. Figures 5 and 6 show a trypan blue assay with the Kelly cell line for the effect of IgM on these cells. The dark cells (blue cells) represent dead cells. From this test it can be seen that the interaction of IgM and complement leads to killing of the tumor cells. Neither IgM alone nor complement alone lead to any significant killing of the cells.

Figure 6 shows a long-term study of IgM cytotoxicity in which eight plasma preparations of an individual donor were collected over a period of time and the cytotoxicity thereof was determined in a Pl assay on Kelly cells. It can be seen from this that the cytotoxicity in the plasmas remained approximately constant over a period of more than 30 months, ie this donor produces approximately equal amounts of cytotoxic IgM.

In summary, the present invention provides the development of new products for cancer therapy from the source of raw material blood plasma. The present invention particularly preferred method of isolating particularly pure and highly concentrated cytotoxic IgM antibody preparations from blood plasma is compatible with the classical plasma fractionation, so that previously unused portions of the blood plasma can now be a profitable and healing use.

The fact that natural IgM antibodies can be used by healthy donors, ie people who are considered to be blood donors, fears the fear of side effects in the background. The polyclonal approach, which is made possible by using the source of raw material blood plasma from blood donation pools, leads to a minimization of the risk of resistance since a large number of immunological targets are recognized. Of course, the present invention is also with the use of monoclonal antibodies or other conventional or immunological Therapies can be combined in tumor patients. The construction of a database allows access to donors with high levels of cytotoxic IgM antibodies.

The invention provides an optimized method for obtaining purified and highly concentrated cytotoxic IgM antibody preparations. The essential steps here are the use of the cytotoxic fractions which are identified from the large number of plasma samples after preferential DEAE adsorption and elution as suitable chromatographic fractions via a selective screening assay. In a second essential step, the IgMs are isolated from this cytotoxic fraction and separated from non-cytotoxic IgMs. The method enables a high purity of the cytotoxic IgM antibody preparations and the lowest possible loss of cytotoxicity compared to the starting materials.

For the characterization of the IgM antibodies they can be detected by anti-IgM antibodies. The cytotoxicity is generally checked by cell culture assays. The invention can be regarded as economically efficient, because in addition to the classic plasma fractionation previously unused side fractions can be developed and can be implemented by the major blood products manufacturers in their processes.

The following examples illustrate the process steps in particularly preferred processes according to the invention:

EXAMPLES

Purification process of human natural cytotoxic IgM antibodies

1. Schematic representation of Aufreiniqunq

Concentrate plasma and

rebuffering

.

DEAE Chromatography pH 8.2 Ultrafiltration DEAE Fraction Pool Ethanol Precipitation 9% pH 5.3 Ultrafiltration Ust 9% Precipitation Ethanol Precipitation 25% pH 7.3

4,

Dissolution of sediments pH 5.9

SEC Chromatography pH 7.3

4,

Final ultrafiltration

Flow chart for the separation of the IgM fraction active in the PI assay 2. Cryoprecipitation and ultrafiltration

2.1. Carrying out cryoprecipitation:

Device: Minifuge / RT

Sample: Plasma

Conditions: 13000 rpm / 10 min / RT

Removal: combine supernatants

2.1. Carrying out ultrafiltration: Device: Amicon cell

Membrane: Pall Omega K30 OM030076 / 30k

Target buffer: 20 mM Tris / 40 mM NaCl / pH 8.2

Procedure: 20 mL US from KP confined to <5 mL + 50 mL buffer

> 55 mL restricted to <5mL

Withdrawal: <5 mL actual volume

Rinse ultrafiltration chamber with 5 mL target buffer

Combine volume to 1 1 mL

Filtration: Filter 1 mL through 0.45 μηη Nalgene syringe filter

Corresponds to the starting material of the DEAE DEAE chromatography

Execution:

Device: AKTA Explorer 100

Column: DEAE CV28ml

Eluent A: 20 mM Tris / 40 mM NaCl / pH 8.2

Eluent B: 20 mM Tris / 250 mM NaCl / pH 8.2

Method: manual implementation at the moment;

Flow: 3 ml / min

Order: 5 ml_ sample loop

Sample: 10 mL of plasma after ultrafiltration / AM DEAE

Step gradient: 10% increments each Addition Eluent B; with visual assessment of

elution

Fractions: 10 ml_

Sampling: AM / DL / fractions / IgM fraction pool (current area)

3.2. chromatogram:

See Figure 2

Ultrafiltration / precipitation 9%

Carrying out ultrafiltration:

Device: Amicon cell

Membrane: Pall Omega K30 OM030076 / 30k

Target buffer: 50 mM phosphate / 100 mM NaCl / pH 5.3 Procedure: 20 mL fraction pool from DEAE Chromatography condense to <5 mL + 50 mL buffer

<55 mL confined to <3 mL

Withdrawal: <3 mL actual volume

Rinse ultrafiltration chamber with 1 mL of target buffer Combine volume

4.2. Carrying out precipitation 9% ethanol:

Precipitation: In the ratio 1365 μΐ sample plus 135 μΐ ethanol (-20 ° C)

Incubation with KS for> 2 hours

Mix gently several times

5. Centrifugation / ultrafiltration / precipitation 25%

5.1. Carrying out centrifugation

Device: Minifuge / RT

Sample: vial from 9% precipitation

Conditions: 13000 rpm / 10 min / RT

Withdrawal: Combine supernatant of vials from 9% precipitation Carrying out ultrafiltration

Device: Amicon cell

Membrane: Pall Omega K30 OM030076 / 30k

Target buffer: 20 mM Tris / 150 mM NaCl / pH 7.3

Procedure: combined supernatants from 9% precipitation

Constrain to <3 mL + 50 mL buffer

<53 mL constrict to <8 mL

Withdrawal: <8 mL actual volume

Rinse ultrafiltration chamber with 1 mL of target buffer Combine volume

5.3. Carrying out precipitation 25% ethanol:

Precipitation: in a ratio of 750 μΐ sample plus 250 μΐ ethanol (-20 ° C)

Incubation with KS for> 3 hours

Mix gently several times

6. Centrifuge and then dissolve the sediments

6.1. Carrying out centrifugation:

Device: Minifuge / RT

Sample: Vial from 25% precipitation

Conditions: 13000 rpm / 10 min / RT Removal: discard supernatants from 25% precipitation

Carrying out the sediments:

Buffer: 0.1 M sodium acetate pH 5.9

Sediments: sediments dissolve in about 500 μΙ / 0.1 M sodium acetate pH 5.9

Incubation with KS for> 3 hours

7. Centrifugation and filtration

7.1. Carrying out centrifugation: Device: Minifuge / RT

Sample: sediments dissolved in 0.1 M sodium acetate pH 5.9

Conditions: 13000 rpm / 10 min / RT

Withdrawal: combine supernatants

7.1. Carrying out filtration:

Filtration: filter through 0.45 μιη Nalgene syringe filters

Corresponds to the starting material of the SEC SEC chromatography

8.1. Execution:

Device: ÄKTA Explorer 100

Column: SEC 1660 Superdex 200

Eluent: 20 mM Tris / 150 mM NaCl / pH 7.2

Method: manual implementation at the moment;

Flow: 1 .5 mL / min

Application: 2 mL sample loop

Sample: 2 mL dissolved IgM sediments

Fractions: 4 mL

Sampling: AM / fractions / IgM fraction pool (current area)

8.2. Procedure: see FIG. 3

9. Ultrafiltration / Conversion to the final product

9.1. Procedure: Device: Amicon cell

Membrane: Pall Omega K30 OM030076 / 30k

Target buffer: 20 mM Tris / 150 mM NaCl / pH 7.3

Procedure: fraction pool from SEC chromatography

constrict to <3 mL

Withdrawal: <3 mL actual volume

Rinse ultrafiltration chamber with 1 mL of target buffer Unite volume

Aliquot the final product

10. Analytics and result

10.1. Methods used for analytics

CBB Assay / PI Bioassay on Kelly cells / Coomassie PAAGE / Western Blot

10.2. Coomassie PAAGE

end product

See Figure 7

10.3. Coomassie PAAGE

Verlaufsqel

See Figure 8

Result: There are gangs in both track 5 and track 8 in the DSP gradient angel

To see IgM altitude.

10.4. Western blot

IgM course

See Figure 9

Result: In the DSP gradient angel in the WB both in lane 5 and in lane 8

IgM detected. 10.5. Western Blot IgM / IgG

end product

See Figure 10

Result: In the final product IgM is detected in the WB

In the final product, no bands with anti IgG AK can be detected in the WB

10.6. Kelly PI Assay

course

See Figure 1 1

11. Used buffers

DEAE: 20mM Tris / 40mM NaCl / pH 8.2

20 mM Tris / 250 mM NaCl / pH 8.2 Precipitation 9%: 50 mM phosphate / 100 mM NaCl pH 5.3

Precipitation 25%: 20 mM Tris / 150 mM NaCl / pH 7.2

Dissolve in front of SEC: 0.1 M sodium acetate pH 5.9

SEC: 20 mM Tris / 150 mM NaCl / pH 7.2 V. Key Publications

Cohn E.J. et al. (1944)

CHEMICAL, CLINICAL, AND IMMUNOLOGICAL STUDIES ON THE PRODUCTS OF HUMAN PLASMA FRACTIONATION. I. THE CHARACTERIZATION OF THE PROTEIN FRACTIONS OF HUMAN PLASMA. J Clin Invest. 4: 417-32.

Bolande, R.P. (1960)

Cytotoxic action of normal human serum on atypical mammalian cell lines Lab. Invest, 9: 475-489

Erttmann et al. (1996)

Naturally occurring humoral cytotoxicity against neuroblastoma (NB) cells in healthy persons and NB patients

Pediatr. Hematol. Oncol. 13: 545-548

David et al. (1996)

Growth arrest of solid human neuroblastoma xenografts in nude rats by natural IgM from healthy humans

Nature Medicine, 2: 686-689

Laban S. (2008)

Sera and plasmas of healthy blood donors completely lyse melanoma cells.

Dissertation Medical Faculty Mannheim of the Ruprecht-Karls-University Heidelberg.

Ollert, M.W. et al. (1996)

Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.

Prc. Natl. Acad. Be. USA 93: 4498-4503.

Ollert, M.W. et al. (1997)

Mechanism of in vivo antineuroblastoma activity of human natural IgM.

Europ. J. of Cancer, 35: 1942-1948

Pool, J.G. (1965)

Preparation and testing of antihemophilic globulin (Factor 8) sources for transfusion therapy in hemophilia. Description of a new sterile concentrate process for blood banks.

Scand J Clin Lab Invest. 1965; 17: Suppl 84: 70-7. Schmitt et al. (1999)

Natural Human IgM Antibodies in the Therapy of Neuroblastoma: Preliminary Results of a Phase I / II Therapy Study.

Klin. Paediatr. 211: 314-318. Schultz et al. (1975)

Detection in colorectal Carcinoma of the human colon and rectum.

Int J Cancer. 1975 Jul 15; 16 (1): 16-23.

Schwartz-Albiez, R. (2007)

Natural anti-tumor cytotoxic antibodies: A novel strategy for tumor therapy Meeting Abstract, Naturally Occurring Antibodies in Health and Disease, Bern, 19.-20. October, 2007, p.23

Schwartz-Albiez et al. (2008)

Cytotoxic natural antibodies against human tumors: an option for anti-cancer immunotherapy?

Autoimmune Rev, 7: 491-495

Tatum AH.

Large scale recovery of biologicaily active IgM (95% pure) from human plasma obtained by therapeutic plasmapheresis.

J Immunol Methods. 1993 Jan 14; 158 (1): 1-4.

Claims

1. Preparation of Cytotoxic IgM Antibody, which is substantially free of non-cytotoxic IgM antibodies and of antibodies of other classes.

2. Preparation of cytotoxic IgM antibodies according to claim 1, wherein cytotoxic IgM antibodies are present therein in a purity of 85% and in particular 90%.

3. Preparation of cytotoxic IgM antibodies obtainable by isolation from blood, serum or plasma of donors, especially healthy donors, in which the liquid blood components are subjected to a first chromatographic separation, chromatographic fractions are assayed for cytotoxicity in a cell culture assay with tumor cells, cytotoxic Fractions are optionally subjected to further purification and separation steps and by rechromatographic separation, a fraction is isolated, which has more than 80%, based on blood plasma constituents, cytotoxic IgM antibody.

4. Preparation according to claim 3, wherein the first chromatographic separation of anion exchange materials, in particular diethylaminoethylcellulose is performed.

5. Preparation according to claim 3 or 4, wherein a size exclusion chromatography (size exclusion chromatography) is carried out as a new chromatographic separation.

6. Preparation according to one of claims 3 to 5, according to which the purification is carried out from blood, plasma or serum of a single donor or a plasma pool.

7. Preparation according to claim 4, according to which the DEAE chromatography is carried out at a pH of 7.8 to 8.8, preferably 8 to 8.5 and more preferably 8.2 to 8.3.

8. Preparation according to one of claims 3 to 7, that one or more ultrafiltration and / or one or more ethanol precipitations are carried out as further purification steps.

9. Preparation according to claim 8, wherein an ethanol precipitation with 7 to 1 1% ethanolic solution at a pH of 4.5 to 6.5 and a further ethanol precipitation in 20 to 30% ethanolic solution at a pH of 5.9 to 8.5 is performed.

10. A process for preparing a preparation of cytotoxic IgM antibodies which is obtainable by isolation from blood, serum or plasma of donors, in particular healthy donors, in which the liquid blood components are subjected to a first chromatographic separation, chromatography fractions on their cytotoxicity in a cell culture assay Tumor cells are examined, cytotoxic fractions optionally subjected to further purification and separation steps and by rechromatographic separation, a fraction is isolated, which has more than 80%, based on blood plasma components, cytotoxic IgM antibody.

1 1. A pharmaceutical composition containing a preparation of cytotoxic IgM antibody which is substantially free of non-cytotoxic IgM antibodies and of antibodies of other classes.

12. Pharmaceutical composition according to claim 1 1 for the treatment of tumor diseases.