Tumour therapeutic agent and process for its preparation.

The present invention relates to a process for the preparation of a therapeutic composition for the treatment of tumor-induced diseases derived from tumor cells as well as said therapeutic compositions.

For the book, The Mechanisms of Tumor Immunity, Gres st eds., John Isyl Sons, Μ. See, 1977, page 196, for example, that tumor-induced diseases can be treated by vaccination with tumor cells that have been modified by freezing and fusion, lyophilization, pressure or homogenization. For the same purpose, sub-translucent fractions or cell extracts have also been used.

However, there is no known vaccine against a tumor disease.

The Applicant has surprisingly discovered that

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Aldehyde-treated lyophilized cells derived from human tumors or cell aggregates thus obtained have the antigens which bind to the monoclonal antibodies described in the specification open to the public inspection of the Alsma Patent; treatment of tumor disease.

The subject matter of the present invention is therefore a therapeutic composition for the treatment of a tumor-caused disease, which contains human cells dried or stabilized by a chemical treatment, which contain antigens that have been bound by the monoclonal antibodies referred to in the patent specification. German

HS 33 29 184.

An advantage of such a therapeutic composition over the use of natural cells is that natural cells are not stable so that they must be primed. Consequently, they cannot be standardized.

It is a further object of the present invention the process of preparing a therapeutic composition for the treatment of a tumor-induced disease characterized by antigen-bearing human tumor cells which are bound by the monoclonal antibodies referred to in the disclosure disclosed for public inspection. of German Patent l^s 33 29 184 are dried or stabilized by chemical treatment and processed to a therapeutic composition to which neuraminidase may be added.

Still further object of the present invention is a process for the preparation of a therapeutic composition for the treatment of a disease caused by a tumor, characterized in that:

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It is only possible to isolate the antigen from human cells by the monoclonal antibody described in the published Descriptive Remedy. This is the end of German Patent RS 33 29 184 and a therapsutic composition to which neuraminidase is possibly added is anticipated.

The possibility of strengthening an Immune response by means of neuraminidase is known by way of the specification open to public inspection of German Patent No.²26 20 649.

By therapeutic composition is meant a composition which may be suitable both for prophylaxis and for the treatment of an already manifest disease.

Cells usable within the framework of the present invention are obtained from tumors according to known cell culture procedures. Preferably, the cells obtained by mechanical or enzymatic means are dried or lyophilized from such cultures, possibly after inactivation with mitomycin C, or treated with a stabilizing agent known to those skilled in the art for monoaldehyde or dialdehyde orgenium tissue. with 1 st 6 carbon, carbon.

Suitable agents for chemical stabilization and fixation belong to the bifunctional compounds in a special manner, or those which contain two functional groups which may react with - that is, crosslink - the functional groups of the biological material. Elss are for example dialdehydes, especially aliphatic dialdehydes of 2 to 8 carbon atoms. For this purpose, however, monoalcansis of 1 to 4 carbon atoms such as formic aldehyde, which may functionally react and also bifunctional immune esters such as

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il subsromidate, isocyanates or isothiocyanates.

may use the material as agents that

Biological agents may also serve so-called tanning agents such as tannin and their derivatives or salts of chromium. Also suitable is sulfo-salicylic acid.

In general, cells are either in the form of cell sets or as individual cells. But antigens or parts resulting from the fragmentation of cells isolated from tumor cells can also be used. Such antigens can be obtained from tumor tissue from patients such as from human tumors that develop in nondeficient animals if separate and use.

The defined antigens are obtained from the tumor cells or their fragmented parts with the monoclonal antibodies referred to in nos. Descriptive Memory published by public inspection of German Patent M^s 33 29 134.

Examples of such antigens are, for example, CA (carcinobrium antigen, J. J. Exp. Med. (1965) 122, 467) or MCA (non-specific crosslinker antigen; J. Immun, (1973) III, 1926), which can be isolated. from tumor cells by immune affinity chromatography. For this purpose the monoclonal antibodies described in Table I of the German Patent Publication No. 34 16 774 are covalently linked as purified proteins to CNBr-activated 5epharose 43 and known antigens of these monoclonal antibodies (CA, MCA) are isolated. ) D-TA colon coccinone cell extracts. The appropriate process is described in Pharmacia Affinity Chromatography, Frinciples and ethods, 12-18 (1979), page 15.

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An antigen obtained by monoclonal antibodies may be used as a therapeutic agent, for example, as an active substance for vaccines against a disease caused by the tumor cells from which the antigen was obtained.

Quality control of a material to be used as a vaccine is performed for example by typing with monoclonal antibodies or by separating the global proteins from cells by electrophoresis on clear polyacrylamide SDS-gsl or isoelectric (first dimension) focusing. sm SDS-polyacrylamide gel electrophoresis (2nd dimension) with subsequent gel staining (silver staining) »

The antigen material in combination with an adjuvant, especially neuraminidase (Descriptive Imam open to the public inspection of German Pztsnte U) is preferred.^s 26 20 649), applied intradermally preferably according to the Schachbrstt vaccination procedure (Cancsr Immunoi, and Immunother. 6, 47-58 (1979), esp. Page 48).

A t = 1 vaccine is preferably employed against certain stages of colon carcinoma growth (Duke C) as well as against other tumors having snigenes or epitopes that are present in the vaccine material. These other tumors are solid tumors, for example, pancreatic carcinoma, lung carcinoma. C of the intestines, sinus carcinoma and vaccination carcinoma can be administered parenterally or orally. Antigens may be dissolved in physiological saline or suspended earlier preferably intradermally with P3S (phosphate-buffered sodium straight solution).

be splicroAs quality criteria for stability

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of the antigen composition of the vaccination material two assays were performed;

a) The Terasaki-IIF (indirect immunofluorescence assay using the tumor cells which are developed in the Terasa · ki microtiter plate assay) with monoclonal antibodies of different specificity. By means of the western assay, membrane secretion of antigens can be measured in intact tumor cells against which a number of monoclonal antibodies are available (Cancer Dstection and Prescription 6, 181-184, 1983). This can cause drastic changes on the DE-TA cell membrane during the culture operation;

(b) Solubilization of whole cell proteins by means of a detergent (Hybridoma 3., 413-421, 1932) as well as subsequent silver stained polyacrylamide gel electrophoresis (Anal. Biochem. 105,

361-363 1930). The combination of these techniques surely ensures that there are no significant changes in the total protein content of the DE-TA cell line.

The following examples clarify the present invention.

Example 1

SVDC carcinoma cell line was cultured in a monolayer cell culture growing in medium

F.PFI-1640 -ί-ioore, E. R, Gemer, RE, Franklin, A., A., Cultures of Normal Human Leucocytes, J, AKA 199, 519-524 (1967), 7 with Fetus Serum ds calf at 10 'in plastic balloons. The confluent cultures of adhesively grown cells were mechanically separated by trypsin stock which

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dissolved in RPRI-164D medium free of fetal calf serum, collogenase was inactivated by addition of calf fetal serum dissolved in RPMI-1640, then the cells were detached from the tissue culture flask and three times with phosphate-compliant sodium chloride (PBS)²Ç.

Ten cells, most of which existed as lumps, were incubated for 1 hour at 37 ° C.^TheC in 1 milliliter of PBS containing 100 micrograms of mitomycin C.

Thereafter, the inactivated cells were then washed three times with PBS and three times with 0.18 molar ammonium bicarbonate buffer which was adjusted to pH 7.4 with acetic acid. A cell pellet corresponding to 10 cells in 100 microliters of the same ammonium bicarbonate buffer was frozen at ~ 70 ° C.²C. The frozen material was then lyophilized and stored in sealed air balloons at -5-42 ° C in a refrigerator. The cell material thus treated may, after resumption in PBS, be employed in a variant vaccination,

(b) incubated for 5 minutes at -42 ° C with 0.1% glutaric aldehyde in PBS, excess glutaric aldehyde was removed by washing three times with PBS, then incubated with 2% BSA (bovine serum albumin) for 5 minutes at -42 ° C was washed three times with PBS. The cells thus treated may be stored at -42 ° C and employed in vaccination of patients. du enrso

c) incubated at 25 ° C^andC in formalin

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7'

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according to Lilly (Benno Romeis (1963), p. 65, chap. 266, Clden burg Verleg, Runique) with eventual shaking at night * Csr.trifugrate cells (about 10), decanting liquid overflowing (1G minutes at SCO g) and the cell pellet was suspended in 7 ml of double distilled water (= wash once). This washing process was repeated four times at 1 hour intervals. The pellet was then washed three times at 1 hour intervals using 7 ml of 70% ethanol each time. Thereafter, the cell pellet was washed three times at 30 minute intervals using 7 ml of ethanol and SOL ds each time. The cell pellet was then washed three times using 7 ml of 96% ethanol each at 30 minute intervals. The cell pellet was then washed three times with 7 ml 99% ethanol each time at 30 minute intervals. Finally, the cell pellet was washed three times with 7 ml of sterile PBS each time at intervals. 30 minutes only was stored under sterile conditions at 42 ° C. Cells can be stored at 42 ° C and patient smprs.

Example 2 as in the

For isolation by antigens by chromatography of the imine affinities of tumor cells, CMBr-activated Sspharose 4Β were covalently linked to purified monoclonal antibodies that unduly react with antigens on tumor cells to be used as vaccine material. Failed according to Pharmacia's book^noAffinity Chromatogrsphy^w, Principies and Rethods, 12-18 (1979), especially page 15. Thereafter, the ligated antibody was incubated in the culture medium with continuous shaking with solubiram obtained from cells from culture flasks by lysis (5 g / l deoxycholate flush 2 hours at 42E cells. These were mechanically released

sodium, 0.5 millimoles / liter of PM3F-phenylmethyl sulfonyl fluoride, PBS, β-0.3), as described in Mybridorna 1,413-421 (1932), especially page 414.

The loaded support was centrifuged and suspended in liss-SDS buffer (20 millimolar pH 8.0 tris-HCl; DKSF 1 millimole / liter; Nonidst P-4Q (= octiphenyl stylene oxide; Fluka AE) i 5 g / l of sodium deoxycholate; 1 millimole / liter of ethylenediaminetstracetate; 1 g / liter of sodium dodecyl sulfate (SDS)) for washing.

This washing process was repeated three times then the support was washed twice in SDS buffer lysis buffer and then once with a wash buffer (2 molar in tris-HCl, pH 8.0 10 millimoles / liter EDTA, 0.5 g / l NP-40). Thus purified antigens were separated from the solid support either by heating for 5 minutes at 95 ° C or by incubation in 6 moles / liter NH SCN for 3D minutes at + 42 ° C.