CA1077840A - Tumor antigen and process for the preparation thereof - Google Patents

Abstract

TUMOR ANTIGEN AND PROCESS FOR THE PREPARATION THEREOF
Abstract 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 re-lates furthermore to immunotherapeutic medicaments against tumor affections which consist of or contain the tumor antigens obtained according to the above process.

Description

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, The present invention relates to -tumor antigens ~hic~ c~n be obtaine~ from tumor cells ~y m~ans of a s~ecial ex~raction process, and a p~ocess for the preparation thereof. The in--vention relates fur-thermore to immunotherapeutic medicaments against tumor affections which consist of or contain the tumor antigens obtained according to the above process.
The immunologic tumor therapy is in principle based on trials to change the antigenic structure of tumor cells a~cord-ing to various methods, for example by treatment with neuramini-dase, in order to make them seem immunologically foreign to the organism to be treated and to incite the latter one to an immuno~
logic answer to the tumor cells.
It has also been proposed to obtain cell-free tumor anti-gens by extraction from tumor cells, in which process there were used potassium chloride solutions, detergents or natural lysolecithin (Davies and Cater, sritish 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 C1 (R~ in formula I) of 16 to 18 and more carbon atoms, synthetic lysolecithin analogs having chain lengths of below 16 carbon atoms are considerably more suitable for the purification of defined antigen fractions, thus obtaining a higher immunity rate and considerably higher yields. Moreover, in contrast to natural lysolecithin, these substances are not degraded by the tumor cells during the purification of the tumor antigen fraction, because they are not affected by the enzyme systems being present in the cells. Furthermore, the short chain synthetic lysolecithin analoys have a much smaller size of micel-~J~

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les, on ~7hich fact ~her~ is obvious:Ly based their hl~hly improve~
capabilit.y of so:lu,bilizing proteins, as compaYed to ].ong~chain lysoleci'chins which form very large micelles (see thesi.s B. Arnold, Zum ~echanismus der Haerno],yse durch Lysolecithin. -Quanti.tative Untersuchungen mit radioakti.v markierten Lysolecithin-Analoga; Freiburg i. Br. 19'75)~
It is therefore the object of the present invention to avoid using natural lysoleci.thin in the preparat,ion of cell-free tumor antigen~ and to prepare antiyens from tumor cells which, after administration to a host, are able to provoke immu~
nity against the tumor.
In accordance with this invention, there is provided a process for preparing tumor antigens by extraction from tumor cells, which comprises treating tumor cells to be obtained in suspension according to known methods w.ith a compound of the formula I, separating subsequently the cell residue from the supernatant and optionally concentrating it to form substances havin~ a sedimentation behavior in the high-speed centrifuge of from S = 3 to S = 16 (Svedberg value~).
Com~ound of formula I:

f H C - O - A

wherein ~1 i,s long-chain alkylcarbonyl or alkoxyl having especial-ly a chain length of 8 to 18 carbon atoms, preferably a chain length of 10 to 14 carbon atoms;
R2 is H or CH3;
R3 is H, OH, alkylcarbonyl or al.ko~yl having a chai ~ 3 ,:~ , , ' ' . ' ' .

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length of 1 to 3 c~bon atom~, or benzyl o~ C113;
R1 and R3 may be interchanyed:
R1 lony-chain substitutec1 = ~ -lysolecithin analog R~ long-chain substituted = ~lysolecithin analog 5 - A is phosphorylcholine, phosphoryl-ethanolamine or phosphorylserine, perferably phosphorylcholine.
Fractions concentrated according to the invention in the high-speed centrifuge can be obtained for e~ample within 1 hour at about 1 x 10~ x g. So]utions of the active fraction(s) are ~Q optically clear and may be filtered without loss of activity by means of filters the pore diameter of which is about ~.22 ~.
The compounds of the formula I may be obtained for example according to Arnold, D., Weltzien, H.U. and O. Westphal;
Vber die Synthese von Lysolecithinen und ihren ~theranaloga;
~5 Liebigs Ann. Chem. 709, 234-239 (1967);
Weltzien, H.U. and O. Westphal;
O-methylierte und O-acetylierte Lysolecithine; Liebigs Ann.
Chem. 709, 240 243 (~967);
Eibl, H. and O. Westphal;
2Q Pa]rnitoyl-propandiol-(1,3~-phosphorylcholin (2-Desoxy-lyso-lecithin und~.~ -Alkandiol-Analoga; Liebigs Ann. Chem. 709, 244-247 (1967).
In a preferred embodiment, tumor cells are obtained and prepared as follows: generally appliable techniques are des-cribed for example in P.F. Kruse, M.K~ Patterson; Tissue Culture;
Academic Press 1973. Cell suspensions are used as starting material which, in the case of suspension tumor, may be obtain-ed in simple manner by distributiny the cells in a suitable

2 medium. Solid tumors are advantageously crushed and brought in-,~ _ -. ' ~

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to the form of a cell suspension by a suitahle en~.na-tic, pr~-ferably proteolytic treatment.
The tumor cell suspension so prepared is suspended in a concentration of preferably 1 - 5 x 107 tumor cells/ml in a physiologically tolerable aqueous solution, preferably a suitable buffer system usual for biochemical operations, for example buffered isotonic saline solution, and subsequentl~, a compound of the formula I is added. The concentration of compound of formula I is from 0.05 to 10 mg/ml, preferably from about 0.2 to 0.7 mg/ml. In case of an especially pronounced hydrophilic character of the compound of formula I, its concentratior in aqueous systems may be increased above 10 mg/ml. The extrac-tion time depends substantially on the concentration of the com-pound of formula I: especially hiyh concentrations require a relatively short extraction time, low concentrations a relatively long one. For the e~traction, the batch is abandoned for a period of from 30 minutes to 20 hours, preferably about 10 to 15 hours, at a temperature below 15 C, preferably from 0 to 5~ C. Advantageously, the batch is slightly moved or 2 a shaken.
~ubsequently, the cell residue is separated from the suspension, advanta~eously by centrifugation. The supernatant solution may then be centrifuged in two steps: first, cells or cell fragments are separated at relatively low speed, and sub-sequently, the supernatant which contains the intended antigen is obtained after high-speed centrifugation of more than 100 000 x g. It has proved to be most advantageous to obtain the intended antigen from a supernatant of a centrifuyation of about 500 000 x ~` g for several hours, preferably about 2 to 6 hours.

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By means o~ known proteochemic~1 m~tho~s, the tumor-actlve antigen fraction is isolated or concentra-ted frorn this centri-~
fuge supernatant. The followiny methods are preferably applied:
molecular sieve filtration, especially on a porous glass carrier, furthermore ion exchange chromatography.
The declsive criterion of aptitude as tumor antigen prepara-tion is the protective action in animal tests, as far as ani-mals are at disposal for the corresponding kind of tumor. If this ~s not the case, fractions may be chosen from antigen pre-1 parations of corresponding tumors. The active fractions may alternatively be chosen by a preliminary thest involving directly the patient, for example by demonstrating that the corresponding fraction can react with antibodies of known tumor specificity.
The reaction may be demonstrated either in vivo by testlng the ~5 skin reaetivity of a patient affected by a determined tumor or n vitro, for example by proving a cytotoxicity due to corre-sponding antibodies.
~he tumor antigens obtainable in this manner are efficient immunotherapeutic medicaments against tumor a~fections.
By immunization of test animals, for example mice, with tumor antigens prepared according to this invention, prophylaxis against a tumor implanted for a test is possible. Most of the animals i~mmunized about 3 to 10 days before implanting the tumor survive this subsequent tumor implantation, while the control animals die without exception.

A therapeutic treatment of tumor-carrying test animals with the tumor antigens aceording to this invention is also possible, in which tests an important reduction of the death rate of the tumor-carry:iny animals is ob~ained.

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Thus, ~he present invention provides ~urthermore irnmuno-therapeutic medicaments ayainst tumor a~fect~ons which consist or contain the tumor antigens obtained according to the process o~ the invention~ These medlcaments are manufactured in a manner usual for parenteral adrninistration. In order to in-crease the immunologic activity, these medicaments may contain suitable additives, for example aluminum hydroxide or lysoleci-thin analogs. Especially suitable are compounds of the formula I, wherein R1 is C1~-alkyl, R2 is H and R3 is OH or OCH3, and A is phosphorylcholine.
The following example illustrate~the invention.
E X A M P L E:
-Tumor cells are obtained from the peritoneal cavity of mice and worked up in the following manner:
~5 10 days after having transplanted the methylcholanthrene-induced tumor to Balb/c mice, the tumor-carrying animals are killed by nitrogen. The peritoneum is laid bare under sterile conditions, the peritoneal cavity is punctured with a needle and the tumor cells are withdrawn. Subsequently, the peritoneal 2Q cavi~y is rinsed twice with 5 ml of buffered saline solution.
The tumor cells are placed into a centrifuge glass con-taining 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 1000 g, and subsequently washed twice with buffered saline solution.
Erythrocytes present in the suspension tumor are lysed by osmotic hypotonic shock treatment. To achieve this, first about 50 ml of icecold 0.2 % saline solution are added to 2~ t~e total cell sediment and, after ~0 seconds, the same volume 7~
of 1.6 % saline solution is added, 50 that a final concentra-tion of 0.9 ~ is attained. By centrifuying the suspenslon batch again at 1000 g, the lysed erythrocytes are separated from the tumor cells which have not been damayed by the hypo-tonic treatment, and these erythrocyte-free tumor cells are then resuspended.
The cells are washed thrice in a phosphate-buffered, sterile 0.1 M saline solution. The cells are resuspended and subsequently adjusted to a concentration of 30 x 1 o6 cells/ml.
The compound of the formula I, wherein R1 is alkoxyl having a chain length of 12 carbon atoms, P2 and R3 each are H and A is phosphorylcholine CH2 - o - C12 25 2 P CH2 - CH2 - N(~) (CH
O (--) 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 suspen- -sion being slightly moved continuously in a tumbler during this period. Agglomerations of the tumor cells possibly occuring are redistributed by more vigorously shaking for a short time.
After having completed the addition of the compound, the follow-ing suspension is obtained: 20 x 106 cells/ml/0.3 mg of compound according to the above formula. The tumor cells are continued to be slightly moved for 20 hours at 4 C. Subse~uently, the ~, .
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tota:L suspension is centri~uged as follo~7s:
1) 20 minwtes at 1000 y. The sediment is rejected.
2) The supernatant of the first centrifugation step is cen-trifuged again at 100 000 x g x h (K = 147). Sediment and supernatant are separated; floated ~at haviny been care-fully suction-filtered before. The supernatant is further worked up. The sediment is resuspended in 1/10 of the starting volume.

3) The supernatan~ 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/lO of the starting volume. Part of the supernatant is centrifuged again as follows:

4) 2.5 x 1o6 x g x h (K = 89 for 14 hours~. Sediment and ~5 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 h and 2.5 x 106 are further separated into high molecular weiyht and low molecular weight amounts in a column with a packing of porous glass beads; a solution of 150 ~g/ml of the above sub-stance dissolved in phosphate-buffered sodium chloride serving for elution.
As tumor antigen, there are used those fractions which correspond to a sedimentation value of from 3 to 16 Svedberg units.
The tumor antigen obtained according to this example is diluted with physiologic saline solution until the intended con~
centration is obtained, so that the antigen amounts indicated 29 in the following Tables 1 and 2 correspor,d to protein values _ g _ 110~, 76/S 00~1 ~ .~'77~
and are obtainecl in a volume of 0.2, ml. The anirnals are given a subcutaneous injection at 4 diferent places. The op-timum moment for starting the prophylactic immuni~ation is frorn day 4 to day 8 before implantation of the methylcholanthren~-induced tumor.
Table 1 shows the survival rate of mice having a methyl-cholanthrene tumor.
T A B L E

Control without tumor S ~g10 ~g 25 ~g 50 ~g 100 ~lg antigen *

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surviving/total number of animals For a therapeutic treatment of (Balb/c x C57Bl)F1 mice with the tumor antigen prepared according to the process des-cribed above and in the Example, the mice were given subcutane-ous injections at 4 different places from the 2nd to the 8th day after implantation of -the methylcholanthrene-induced tumor, using the amounts of antigen per animal indicated in Table 2~

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Fractions 1 x 106 s 1 x 106 su 2.5 x 1o6 s 2.5 x 1o6 su 0.5 mg 0.1 mg0~5 mg O. I mg/mous~
.. .. .. . . ~ . _ . . _ . . _ 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 ac-celeration = Control without tumor antigen: 0/5 The products of the invention are efficient in the preven~
tive and therapeutic trea-tment of other test animals which carry different tumors as well. In order to demonstrate this, the following tumor tests have been made in addition:
1. Ehrlich ascites tumor in NMRI mice immunized with the puri-fied Ehrlich asci~es tumor cell antigen.
2. Myeloma X 5563 in C3H mice, immunized with the purified myeloma X 5563 tumor cell antigen.
3. MPC11 myeloma in Balb/c mice, immunized with the purified MPC1~ myeloma tumor cell antigen.
4, Lewis lung tumor in C57Bl mice, immunized with the purified Lewis lung tumor cell anitgen.
Preparation and purification of the corresponding antigens are carried out in analogy to the process of the invention and the above Example,

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a tumor antigen in which tumor cells are treated with a compound of the formula I

I

wherein R1 is long-chain alkylcarbonyl or alkoxyl having a chain length of 8 to 18 carbon atoms;
R2 is H or CH3;
R3 is H, OH, alkylcarbonyl or alkoxyl having a chain length of 1 to 3 carbon atoms, or benzyl or CH3;
R1 and R3 may be interchanged and, A is phosphorylcholine, phosphoryl-ethanolamine or phosphorylserine, in order to obtain a suspension, and the cell residue of the resultant suspension is separated from the supernatant and the supernatant is recovered.

2. A process as claimed in claim 1 in which A is phosphoryl-choline.

3. A process as claimed in claim 1 in which the supernatant is concentrated to form substances having a sedimentation behaviour in the high speed centrifuge of from S = 3 to S = 16 wherein S
represents Svedbery values.

4. A tumor antigen, whenever obtained according to a process as claimed in claim 1, claim 2 or claim 3 or by an obvious chemical equivalent thereof.

5. A process as claimed in claim 1 in which the suspension contains tumor cells in a concentration of 1 to 5 x 107 tumor cells/ml of solution.

6. A process as claimed in claim 5 in which the tumor cells are in suspension in a buffered isotonic saline solution.

7. A process as claimed in claim 1 in which the cell residue is separated from the supernatant by centrifugation.

8. A tumor antigen, whenever obtained according to a process as claimed in claim 5, claim 6 or claim 7 or by an obvious chemical equivalent thereof.