IE44943B1

IE44943B1 - Tumor antigen and process for the preparation thereof

Info

Publication number: IE44943B1
Application number: IE887/77A
Authority: IE
Original assignee: Max Planck Gesellschaft
Priority date: 1976-05-04
Filing date: 1977-05-03
Publication date: 1982-05-19
Also published as: CA1077840A; DK194477A; ZA772648B; BE854269A; GB1575545A; JPS52134011A; ATA313277A; AT362497B; AU2481877A; NL7704724A; NZ183980A; FR2391733B1; IE44943L; IL51989A0; IL51989A; AU517395B2; DE2619715A1; SE7705070L; IT1074325B; LU77245A1

Abstract

TUMOR ANTIGEN AND PROCESS FOR THE PREPARATION THEREOF of the disclosure: The present invention relates to tumor antigens which can be obtained from tumor cells by treating tumor cells with a short chain synthetic lysolecithin analog, separating the cell residue and recovering the supernatant; the invention relates furthermore to immunotherapeutic medicaments against tumor affections which consist of or contain the tumor antigens obtained according to the above process.

Description

The present invention relates to tumor antigens which can be obtained from*1 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 cell-free 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 seq.).

It has now been found that, surprisingly, in contrast to the natural lysolecithin having undefined chain lengths in (R^ in formula 1) 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, Sum Mechanismus der Haemolyse durch Lysolecithin.Quantitative Untersuchungen mit radioaktiv markiarten Lysolecithin Analoga· Freiburg i. Br. 1375).

The present invention accordingly provides a process for preparing rumour antigens, which comprises treating a suspension of tumour cells with a compound of the general formula Σ H G-R Ί R2"CR3 . ’ HjC -0-A in which A represents a phosphoryl chol ine, phosphorylethsnolamine or phosphorylserine group: R represents a long chain alkylcarbonyl or alkoxy group having from S to 18 carbon atoms, especially IQ to 14 carbon atoms; Ro represents a hydrogen atom or a methyl group? R_ represents a hydrogen atom, a hydroxyl group or an 3 alkylcarbonyl or alkoxy group having from 1 to 3 carbons, or represents a benzyl or methyl group; R 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 R^ is the long chain group are analogues of a-lysolecithin, whereas those in which R3 is the long chain group are β-lysolecithin analogues.

Especially preferred are those compounds of formula I in which R, represents a C, -alkoxy or C, -alkylcarbonyl group, R„ J- 18 io 2 represents a hydrogen atom, R.„ represents a hydroxyl group or a methoxy group, and A represents a phosphorylcholine group.

The 3 to 1SS fractions are obtained by high speed centrifugation, for example, at about 1 x 10° 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 O.22|i.

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 and ihren Atheranaloga; Liebigs Ann. Chem. 709, 234—239 (1967); Weltzein, H.U. and 0. Westphal; O-methylierte and 0-acetylierte Lysolecithine; Liebigs Ann. Chem. 709, 240—243 (1967); Eibl, H. and 0. Westphal; Palmitoyl-propandiol—(1,3)—phosphoryleholin (2-Desoxy-Lysolecithin und eo.cu-Alkandiol-Analoga; Liebigs Ann. Chem. 709, 244— 247 (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 χ 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 ϊ is added. The concentration of the compound of formula I is 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 hydrophilic 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 tha extraction, the mixture is allowed to stand, for example, for a period of from 30 minutes tc 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 centrifugation. The centrifugation may ba carried out in two or more steps: first, the cells and/or cells fragments are separated at a relatively low speed, that is to say, at less than 100,OOOg and, subsequently, the supernatant, which contains the desired antigen, is obtained after high-speed centrifugation at 100,OOOg or more. It has proved to be most advantageous to obtain the desired antigen from the supernatant by centrifugation at about 500,OOOg 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 10gg and 2.5 x 10g g.

By means of known proteochemical methods, the tumouractive antigen fractiori 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 that 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 immunised 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 4·4 3 4 3 the process of the invention in admixture or conjunction with a pharmaceutically suitable carrier. These preparations are in a form suitable for parenteral administration. Tn 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 Patent Specification No. 44927 , ' Especially suitable for compounds of the formula I, wherein R. is C_ -alkoxy X X» or Cj.g-alkylcarbonyl, Is H and R3 Is 0K or and A is phos phoryIcholine.

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 transplated into a number of Balb/c 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 centrifuge 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 4°C and lOOOg. 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 ice cold 0.2% 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 treamtment, and these erythrocyte-free tumour cells are then resuspended. t The cells are washed thrice in a phosphate-buffered, sterile Θ.1Κ saline solution. The cells are resuspended and subsequently 6 adjusted to a concentration of 30 x 10 cells/ml. The compound of the formula I, wherein is alkoxyl having a chain length of 12 carbon atoms, Rg an<^ ^3 eaca are H and A is phosphorylcholine Ii (+1 C02-0-|-CH2-CH2-4?'‘ (CH3)3 0(-) 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 imte. After having completed the addition of the compound, the following suspension is obtained: 20 x 10 cells ml/0.3 mg of compound according to the above formula. The tumour cells are continued to be slightly, moved for 20 hours at 4°C. Subsequently, the total suspension is centrifuged as follows: - 9 1) 20 minutes at lOOOg. 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 x 10^ 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 χ 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 fhe volume.

The supernatants of the centrifugations 1 x 10^ x g x h and 2.5 x 106 are further separated into high molecular weight and lov; 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 phosphate-buffered 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 a 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 methylcholanthrene-indueed tumour.

TABLE 1 Control without tumour antigen 5gg 10gg 25 gg 50gg lOOgg 0/5 4/5 4/5 4/5 4/5 3/5 * surviving/total number of animals TABLE 2'* Fractions 1 X io6 S 0.5 mg 1 x io6 SU 0.1 mg 2.5 x 106 S 0,5 mg 2.5 x 10δ SU 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. έ¥ = Control without tumour antigen: 0/5 Table 1 shows the survival· rate of mice having a mefchylcholanthrene tumour.

For a therapeutic treatment of (Balb/c x C57B1) , mice v/ith Pl 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 mathylcholanthrena-induced tumour, using the amounts of antigen par animal indicated in Table 2.

The products of the invention are efficient in the preventive and therapeutic treatment in 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 EfMRI mice immunized with the purified Ehrlich ascites tumour cell antigen. 44343 2. Myeloma X 5563 in C3H mice, immunized with the purified myeloma X 5563 tumour ce,ll antigen. 3. MPCll myeloma in Balb/c mice, immunized with the purified MPCll myeloma tumour cell antigen. 4. Lewis lung tumour in C57B1 mice, immunized with tha 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.

Claims

CLAIMS:

1. A process for preparing tumour antigens, which comprises treating a suspension of tumour cells with a compound of the general formula I H C-R 2 | 1 R--C-R (I) h 2 c -G,-A in which A represents a phosphorylcholine, phosphorylethanolamine or phosphorylserine group; R^ represents a long chain alkylcarbonyl or alkojcy group having from 8 to 18 carbon atoms; R^ represents a hydrogen atom or a methyl group; R^ 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; R^ and R^ being interchangeable; separating the supernatant from fche 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 R^ 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 R^ represents a C-alkoxy or C^g-alkylcarbonyl group, R^ represents a hydrogen atom, R^ 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 * Ο 4 J an alkoxy group having 12 carbon atoms, R^ ant ^ R 3 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 5 of 1-5 χ 10 7 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 concentra10 tion 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 15 the resulting supernatant is centrifuged at 100,000 g or more.

11. natant is

12. natant is

13. natant is A process as claimed in claim 10, wherein the superfirst centrifuged at below 100,OOOg. A process as claimed in claim 10, wherein the supercentrifuged at 500,000 g. A process as claimed in claim 10, wherein the supersubjected to a series of centrifugations.

14. A process 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 25 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 14 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, 5 wherein the adjuvant is aluminium hydroxide.

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

21. A pharmaceutical preparation as claimed in claim 20, wherein the lysolecithin analogue is a compound of the general 10 formula I as defined in any one of claims 1 to 5.

22. A method which comprises administering to a nonhuman animal a tumour antigen as claimed in claim IS to prevent and/or treat a tumour.

23. A method as claimed in claim. 22, wherein a lysolecithin 15 analogue is also administered.