GB1575545A

GB1575545A - Tumour antigen and process for the preparation thereof

Info

Publication number: GB1575545A
Application number: GB18430/77A
Authority: GB
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 1976-05-04
Filing date: 1977-05-03
Publication date: 1980-09-24
Also published as: NL7704724A; ATA313277A; IE44943L; IE44943B1; NZ183980A; AU517395B2; AT362497B; IL51989A0; DE2619715A1; ZA772648B; FR2391733A1; CA1077840A; DK194477A; BE854269A; AU2481877A; JPS52134011A; LU77245A1; IT1074325B; IL51989A; SE7705070L

Description

(54) TUMOUR ANTIGEN AND PROCESS OF THE PREPARATION THEREOF (71) We, MAX-PLANcKGesellschaft zur Förderung der Wissenschaften e.V., a body corporate organised according to the laws of the Federal Republic of Germany, of 10 Bunsenstrasse, 340 Göttingen, Federal Republic of Germany, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to tumour antigens which can be obtained from tumour cells, and a process for the preparation thereof.

One method of immunological tumour therapy is based on the principle of changing the antigenic structure of tumour cells in order to make them seem immunologically foreign to the host organism and to incite the organism to an immunologic response to the tumour cells. Neuraminidase is one of the agents used in this method.

It has also been proposed to obtain cellfree tumour antigens by extraction from tumour cells, for which process there have been proposed potassium chloride solutions, detergents, and natural lysolecithin (Davies and Cater, British Journal Experimental Path., 1973, vol 54 p. 583 et sex.).

It has now been found that, surprisingly, in contrast to the natural lysolecithin having undefined chain lengths in C1 (R1 in formula I) of 16 to 18 and more carbon atoms, synthetic lysolecithin analogues, especially those having chain lengths of below 16 carbon atoms, are considerably more suitable for the purification of defined antigen fractions, giving higher yields and ultimately a higher immunity rate on administration. Moreover, in contrast to natural lysolecithin, these substances are not degraded by the tumour cells during the purification of the tumour antigen fraction, because they are not affected by the enzyme systems present in the cells. Furthermore, the synthetic lysolecithin analogues have a much smaller micelle size, which appears to be the reason for their particularly high capacity for solubilising proteins compared with natural lysolecithins, which form very large micelles (see thesis B.

Arnold, Zum Mechanismus der Haemolyse durch Lysolecithin.-Quantitative Untersuchungen mit radioaktiv markierten Lysolecithin Analoga; Freiburg i. Br. 1975).

The present invention accordingly provides a process for preparing tumour antigens, which comprises treating a suspension of tumour cells with a compound of the general formula I

in which A represent a phosphorycholine, phosphorylethanolamine or phosphorylserine group; R1 represents a long chain alkylcarbonyl or alkoxy group having from 8 to 18 carbon atoms, especially 10 to 14 carbon atoms; R2 represents a hydrogen atom or a methyl group; R3 represents a hydrogen atom, a hydroxyl group or an alkylcarbonyl or alkoxy group having from 1 to 3 carbons, or represents a benzyl or methyl group; R1 and R3 being interchangeable; separating the supernatant from the resulting cell residue and, if desired, isolating the fraction of the supernatant having a sedimentation coefficient of 3-16S (Svedberg units).

Those compounds in which R1 is the long chain group are analogues of a-lysolecithin, whereas those in which R3 is the long chain group are ss-lysolecithin analogues.

Especially preferred are those compounds of formula I in which K1 represents a C1 s- alkoxy or C18-alkylcarbonyl group, R2 represents a hydrogen atom, R3 represents a hydroxyl group or a methoxy group, and A represents a phosphorylcholine group.

The 3 to 16S fractions are obtained by high speed centrifugation, for example, at about 1 x 106 g for an hour. The active fraction(s) are optically clear solutions and may be filtered without loss of activity by means of filters having a pore diameter of about 0.22u.

The compounds of formula I may be obtained, for example, according to Arnold, D., Weltzein, H.U. and 0. Westphal; Uber die Synthese von Lysolecithinen und ihren Atheranaloga; Liebigs Ann. Chem. 709, 234239 (1967); Weltzein, H.U. and 0. Westphal; O-methylierte and O-acetylierte Lysolecithine; Liebigs Ann. Chem. 709, 240-243 (1967); Eibl, H. and 0. Westphal; Palmitoyl-propandiol-(1,3)-phosphorylcho lin (2-Desoxy-lysolecitliin und w ,w-Alkan- diol-Analoga; Liebigs Ann. Chem. 709, 244247 (1967).

Generally applicable techniques for carrying out the process of the invention are described for example, in P.F. Kruse, M.K.

Patterson; Tissue Culture; Academic Press 1973. A preferred method is as follows: Cell suspensions are used as the starting material and, in the case of suspension tumour, may be obtained in a simple manner by distributing the cells in a suitable medium. A solid tumour is advantageously crushed and brought into the form of a cell suspension by a suitable enzymatic, preferably proteolytic, treatment.

The tumour cell suspension so prepared is preferably brought to a concentration of 1 to 5 x 107 tumour cells/ml in a physiologically tolerable aqueous solution, preferably a suitable buffer system usual for biochemical operations, for example, buffered isotonic saline solution, and subsequently, a compound of the formula lis added. The concentration of the compound of formula lis generally from 0.05 to 10 mg/ml, preferably from about 0.2 to 0.7 mg/ml. In case of a compound of formula I having an especially pronounced hydro philip character, however, its concentration in the aqueous system may be increased above 10 mg/ml. The extraction time depends substantially on the concentration of the compound of formula I: high concentrations require a relatively short extraction time, low concentrations a relatively long one. For the extraction, the mixture is allowed to stand, for example, for a period of from 30 minutes to 20 hours, preferably about 10 to 15 hours, generally at a temperature below 15"C, preferably from 0 to 5"C. Advantageously, the batch is slightly moved or shaken.

Subsequently, the cell residue is separated from the suspension, advantageously by centrfugation. The centrifugation may be carried out in two or more steps: first, the cells and/or cell fragments are separated at a relatively low speed, that is to say, at less than 100,000g and, subsequently, the supernatant, which contains the desired antigen, is obtained after high-speed centrifugation at 100,000g or more. It has proved to be most advantageous to obtain the desired antigen from the supernatant by centrifugation at about 500,000g for several hours, preferably about 2 to 6 hours. A series of several centrifugations may be carried out, for example, at 100,000 g, 1 x 106g and 2.5 x 106 g.

By means of known proteochemical methods, the tumour-active antigen fraction may be isolated and/or concentrated from the centrifuged supernatant. The following methods are preferably used: molecular sieve filtration, especially on a porous glass carrier and ion exchange chromatography.

The decisive criterion of suitability of a fraction as a tumour antigen preparation is its protective action in animal tests, as far as animals are available for ihat type of tumour.

If this is not the case, fractions may be chosen from antigen preparations of corresponding tumours. The active fractions may alternatively be determined by a preliminary test involving the patient for example, by demonstrating that a certain fraction can react with antibodies of known tumour specificity. The reaction may be demonstrated either in vivo by testing the skin reactivity of a patient affected by a particular tumour or in vitro, for example, by demonstrating a cytotoxicity due to corresponding antibodies.

The tumour antigens obtainable in this manner are efficient immunotherapeutic agents against tumours.

By immunization of test animals, for example mice, with tumour antigens prepared according to this invention, prophylaxis against a tumour implanted for test purposes may be achieved. Most of the animals immunized about 3 to 10 days before implanting a tumour survive the subsequent tumour implantation, while the control animals die without exception.

Therapeutic treatment of tumour-carrying test animals with the tumour antigens according to this invention is also possible, in which tests a significant reduction in the death rate of the tumour-carrying animals is observed.

Thus, the present invention also provides pharmaceutical preparations suitable for use in treatment and prophylaxis of tumours, which comprise a tumour antigen obtained according to the process of the invention in admixture or conjunction with a pharmaceutically suitable carrier. These preparations are in a form suitable for parenteral administration.

In order to increase the immunological activity, the preparations may also comprise suitable additives, for example, aluminium hydroxide or lysolecithin analogues, for example, of the general formula I. Particularly useful are the lysolecithin analogues described and claimed in our Co-pending Application No. 18431/77 (HOE 76/S 005). Especially suitable are compounds of the formula I, wherein R1 is C18alkoxy or C18 -alkylcarbonyl, R2 is H and R3 is OH or OCH3, and A is phosphorylcholine.

The invention also provides a method wherein a non-human animal is administered a compound of the general formula I for the prevention or treatment of a tumour.

The following Example illustrates the invention.

EXAMPLE Tumour cells are obtained from the peritoneal cavity of mice and worked up in the following manner: The methylcholanthrene-induced tumour is transplanted into a number of Baltic mice. 10 days later, the tumour-carrying animals are killed by nitrogen. The peritoneum is laid bare under sterile conditions, the peritoneal cavity is punctured with a needle and the tumour cells are withdrawn. Subsequently, the peritoneal cavity is rinsed twice with 5 ml of buffered saline solution.

The tumour cells are placed into a centi fuge glass containing a volume of about 100 ml of buffered saline solution, in order to prevent precipitation of fibrin in the exudate.

The cells are centrifuged for 5 minutes at 40C and 1000 g, and subsequently washed twice with buffered saline solution.

Erythrocytes present in the suspension tumour are lysed by osmotic hypotonic shock treatment. To achieve this, first about 50 ml of icecold 0.2to saline solution are added to the total cell sediment and, after 40 seconds, the same volume of 1.6% saline solution is added, so that a final concentration of 0.9% is attained. By centrifuging the suspension batch again at 1000 g, the lysed erythrocytes are separated from the tumour cells which have not been damaged by the hypotonic treatment, and these erythrocyte-free tumour cells are then resuspended.

The cells are washed thrice in a phosphatebuffered, sterile 0.1M saline solution. The cells are resuspended and subsequently adjusted to a concentration of 30 x 106 cells/ml. The compound of the formula I, wherein R1 is alkoxyl having a chain length of 12 carbon atoms, R2 and R3 each are H and A is phosphorylcholine is dissolved under sterile conditions in phosphate-buffered saline solution and adjusted to exactly half the volume of the cell suspension. Subsequently, the dissolved compound is added in portions within 60 minutes in an ice bath; the total suspension being slightly moved continuously in a tumbler

during this period. Agglomerations of the tumour cells possibly occurring are redistributed by more vigorously shaking for a short time.

After having completed the addition of the compound, the following suspension is obtained: 20 x 106 cells m1/0.3 mg of compound according to the above formula. The tumour cells are continued to be slightly moved for 20 hours at 40C. Subsequently, the total suspension is centrifuged as follows: 1) 20 minutes at 1000 g. The sediment is rejected.

2) The supernatant of the first centrifugation step is centrifuged again at 100,000 x g x h (K = 147). Sediment and supernatant are separated; floated fat having been carefully suction-filtered before. The supernatant is further worked up. The sediment is resuspended in 1/10 of the starting volume.

3) The supernatant is again centrifuged for 5 hours at 1 x 106 x g x h (K = 78 during 5 hours). Supernatant and sediment are separated and the sediment is resuspended in 1/10 of the starting volume. Part of the supernatant is centrifuged again as follows: 4) 2.5 x 106 x g x h (K = 89 for 14 hours).

Sediment and supernatant are separated, and the sediment is resuspended, as described, in 1/10 of the volume.

The supernatants of the centrifugations 1 x 106 x g x hand 2.5 x 106 are further separated into high molecular weight and low molecular weight amounts in a column with a packing of porous glass beads; a solution of 150 ug/ml of the above substance dissolved in phosphatebuffered sodium chloride serving for elution.

As tumour antigen, there are used those fractions which correspond to a sedimentation value of from 3 to 16 Svedberg units.

The tumour antigen obtained according to this example is diluted with physiologic saline solution until the intended concentration is obtained, so that the antigen amounts indicated in the following Tables 1 and 2 correspond to protein values and are obtained in a volume of 0.2 ml. The animals are given a subcutaneous injection at 4 different places. The optimum moment for starting the prophylactic immunization is from day 4 to day 8 before implantation of the methylcholanthiene-induced tumour.

TABLE 1 Control without tumour antigen Sug 10,ug 2511g 50,ug l00ug 0/5* 4/5 4/5 4/5 4/5 3/5 * surviving/total number of animals TABLE 2* Fractions 1 x 106 S 1 x 106 SU 2.5 x 106 S 2.5 x 106 St; 0.5 mg 0.1 mg 0.5 mg 0.1 mg/mouse day + 2 2/5 2/5 1/5 1/5 day + 4 0/5 1/5 1/5 0/5 day + 6 3/5 1/5 3/5 2/5 day +8 1/5 3/5 2/5 1/5 S = Sediment SU = Supernatant at the corresponding high-speed centrifuge acceleration.

* = Control without tumour antigen: 0/5 Table 1 shows the survival rate of mice having a methylcholanthrene tumour.

For a therapeutic treatment of (Balb/c x C57B1)F1 mice with the tumour antigen prepared according to the process described above and in the Example, the mice were given subcutaneous injections at 4 different places from the 2nd to the 8th day after implantation of the methylcholanthrene-induced tumour, using the amounts of antigen per animal indicated in Table 2.

The products of the invention are efficient in the preventive and therapeutic treatment of other test animals which carry different tumours as well. In order to demonstrate this, the following tumour tests have been made in addition: 1. Ehrlich ascites tumour in NMRI mice immunized with the purified Ehrlich ascites tumour cell antigen.

2. Myeloma X 5563 in C3H mice, immunized with the purified myeloma X 5563 tumour cell antigen.

3. MPCl 1 myeloma in Balb/c mice, immunized with the purified Mac 11 myeloma tumour cell antigen.

4. Lewis lung tumour in C57B1 mice, immunized with the purified Lewis lung tumour cell antigen.

Preparation and purification of the corresponding antigens were carried out in analogy to the process of the invention and the above Example and similar results were obtained.

WHAT WE CLAIM IS: 1. A process for preparing tumour antigens, which comprises treating a suspension of tumour cells with a compound of the general formula I

in which A represents a phosphorylcholine, phosphorylethanolamine or phosphorylserine group; R1 represents a long chain alkylcarbonyl or alkoxy group having from 8 to 18 carbon atoms; R2 represents a hydrogen atom or a methyl group; R3 represents a hydrogen atom, a hydroxyl group or an alkylcarbonyl or alkoxy group having from 1 to 3 carbons, or represents a benzyl or methyl group; R1 and R3 being interchangeable; separating the supernatant from the resulting cell residue and, if desired, isolating the fraction of the supernatant having a sedimentation coefficient of from 3 to 16 S.

2. A process as claimed in claim 1, wherein R1 has a chain length of 10 to 14 carbon atoms.

3. A process as claimed in claim 1 or claim 2, wherein A represents a phosphorylcholine group.

4. A process as claimed in claim 1, wherein R1 represents a Cl8-alkoxy or C18-alkylcar- bonyl group, R2 represents a hydrogen atom, R3 represents a hydroxyl group or a methoxy group and A represents a phosphorylcholine group.

5. A process as claimed in claim 1, wherein R, represents an alkoxy group having 12 carbon atoms, R2 and R3 each represents a hydrogen atom and A represents a phosphorylcholine group.

6. A process as claimed in any one of claims 1 to 5, wherein the tumour cell suspension is brought to a concentration of 1-5 x 107 tumour cells/ml.

7. A process as claimed in any one of claims 1 to 6, wherein the concentration of the compound of formula I in the cell suspension is from 0.05 to 10 mg/ml.

8. A process as claimed in claim 7, wherein the concentration is from 0.2 to 0.7 mg/ml.

9. A process as claimed in any one of claims 1 to 8, wherein the suspension is allowed to stand at a temperature of below 15"C.

10. A process as claimed in any one of claims 1 to 9, wherein the resulting supernatant is centrifuged at 100,000 g or more.

11. A process as claimed in claim 10, wherein the supernatant is first centriifuged at below 100,000g.

12. A process as claimed in claim 10, wherein the supernatant is centrifuged at 500,000 g.

13. A process as claimed in claim 10, wherein the supernatant is subjected to a series of centrifugations.

14. A prdcess as claimed in any one of

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. TABLE 2* Fractions 1 x 106 S 1 x 106 SU 2.5 x 106 S 2.5 x 106 St; 0.5 mg 0.1 mg 0.5 mg 0.1 mg/mouse day + 2 2/5 2/5 1/5 1/5 day + 4 0/5 1/5 1/5 0/5 day + 6 3/5 1/5 3/5 2/5 day +8 1/5 3/5 2/5 1/5 S = Sediment SU = Supernatant at the corresponding high-speed centrifuge acceleration. * = Control without tumour antigen: 0/5 Table 1 shows the survival rate of mice having a methylcholanthrene tumour. For a therapeutic treatment of (Balb/c x C57B1)F1 mice with the tumour antigen prepared according to the process described above and in the Example, the mice were given subcutaneous injections at 4 different places from the 2nd to the 8th day after implantation of the methylcholanthrene-induced tumour, using the amounts of antigen per animal indicated in Table 2. The products of the invention are efficient in the preventive and therapeutic treatment of other test animals which carry different tumours as well. In order to demonstrate this, the following tumour tests have been made in addition: 1. Ehrlich ascites tumour in NMRI mice immunized with the purified Ehrlich ascites tumour cell antigen. 2. Myeloma X 5563 in C3H mice, immunized with the purified myeloma X 5563 tumour cell antigen. 3. MPCl 1 myeloma in Balb/c mice, immunized with the purified Mac 11 myeloma tumour cell antigen. 4. Lewis lung tumour in C57B1 mice, immunized with the purified Lewis lung tumour cell antigen. Preparation and purification of the corresponding antigens were carried out in analogy to the process of the invention and the above Example and similar results were obtained. WHAT WE CLAIM IS:

1. A process for preparing tumour antigens, which comprises treating a suspension of tumour cells with a compound of the general formula I

14. A prdcess as claimed in any one of

claims 1 to 13, wherein the tumour antigen fraction is isolated and/or purified.

15. A process as claimed in claim 1, carried out substantially as described in the Example herein.

16. A tumour antigen, whenever prepared by a process as claimed in any one of claims 1 to 15.

17. A pharmaceutical preparation which comprises a tumour antigen as claimed in claim 16 in admixture or conjunction with a pharmaceutically suitable carrier.

18. A pharmaceutical preparation as claimed in claim 17, which also comprises an adjuvant.

19. A pharmaceutical preparation as claimed in claim 18, wherein the adjuvant is aluminium hydroxide.

20. A pharmaceutical preparation as claimed in any one of claims 17 to 19, which also comprises a lysolecithin analogue.

21. A pharmaceutical preparation as claimed in claim 20, wherein the lysolecithin analogue is a compound of the general formula I as defined in any one of claims 1 to 5.

22. A method which comprises administering to a non-human animal a tumour antigen as claimed in claim 16 to prevent and/or treat a tumour.

23. A method as claimed in claim 22, wherein a lysolecithin analogue is also administered.