NO751769L - - Google Patents

Description

"Polypeptide antigen associated with cancer and method for its production"

The present invention concerns the problem of isolating an antigen linked to canes, which shows monospecificity and is close to

present in many different types of human cancers with different localizations.

Many attempts have been made to demonstrate the presence of tumor reactive antibodies in sera obtained from animals to which preparations of human cancer tumors have been administered. If such experiments should prove to be unambiguously reproducible, this would point to the presence in human cancer tissue of important antigens that are not close to being present in normal tissue, and this should thus conceivably lead to a better understanding of the nature of the neoplastic process.

In order to gain a better understanding of cancer diseases, human cancer tissue has been studied to find out whether there are mono-specific antigens associated with cancer and, above all, to find a reproducible way of purifying and isolating such an antigen. The presence of antigens associated with adenocarcinoma in the colon and digestive system has been demonstrated by Gold et al J. expt. With. 121 (1965) 439-462 and J. expt. With. 122, 1965, 467-487. In these reports, reference is made to previous work by B. Bjørklund, which points to the occurrence of antigens linked to human cancers that are common to the majority of all known malignant tumors of epithelial origin. Boarding school. Arch. Allergy and Appl. Immunol. 1966, 8, 179 and Internat. Arch. Allergy and Apll. Immunol. 1958, 12, 241. In US patent document 3,663,684, a carcinoma embryonal antigen is described which exhibits so-called "CBA— Activity". However, this antigen differs completely from the antigen according to the present invention, which will be shown in detail below in the present description.

The practical and technically applicable isolation of antigen as such, as well as its association with various carinornatic diseases, has so far not been achieved. Thus far, it has not been possible to isolate and characterize antigens linked to human cancers by practical and reproducible methods, nor has it been possible to determine or demonstrate the presence of the antigen in the blood of people suffering from hidden or overt cancer by means of a diagnostic test suitable for large-scale investigations.

In order to make full use of the presence of antibodies specific to tumor-associated antigens in animal antisera as a diagnostic tool, a test must be prepared that shows the presence of the tumor antigen in the patient's blood. The methods that have been proposed so far have not proven effective when it comes to trying to make a certain diagnosis with regard to the presence of Caneer's disease.

One of the main purposes of the present invention is to arrive at a method for isolating and purifying antigen that is linked to cancer, from human tumor tissue or other tissue that contains such antigen.

Another purpose of the invention is the characterization of a

such antigen after its isolation.

A further object of the invention is to provide diagnostic test methods which are based on the use or presence of an antigen associated with human cancer.

A further object of the invention is to arrive at a method for the production of antibodies which are specific for the aforementioned polypeptide antigens which are linked to cancers.

A further object of the invention is to produce a preparation which is usable as an immunization agent.

Another object of the invention is to arrive at a preparation containing such an antigen and a stabilizing amount of albumin in mixture with it and a method for producing such a preparation.

A further object of the invention is to arrive at a diagnostic preparation which contains a particulate material which is labeled with albumin-stabilised antigen.

In the present description including the patent claims, the term "cancer-associated polypeptide antigen" is abbreviated to CAPA.

According to the invention, i.a. practical procedures fon

a) Purification, isolation, characterization and confirmation of the identity and specificity of CAPA; b) Application of such a CAPA for diagnosing the presence of cancer by detecting the presence of circulating CAPA, and c) Method for producing antibodies which are monospecific with respect to such CAPA.

Other purposes and characteristics of the invention will become apparent from the following description of the invention in general terms as well as from specific examples.

A material that has CAPA activity is isolated and purified by the method according to the present invention by homogenizing malignant tissue from autopsies, whereby carcinoma tissue of different types and from different locations are brought together to form a tumor pool. An alternative starting material is tissue from the human placenta or cultures of malignant cells in vitro (for details regarding such cultures see B. BjOrklund, V. BjOrklund and I. Hedlof, J. Nat. Cancer Inst. 261533-545, 1961, "Antigenicity of Pooled Human Malignant and Normal Tissues by Cytoimmunological Technique. III. Distribution of Tumor Antigen).Normal tissue is also collected in parallel with malignant tissue, and in order to achieve a usable sequence of procedure steps leading to Isolation of a pure antigen, a new test procedure, consistent with the accompanying Fig. 1. Thus, the application of biochemical separation methods was guided by a sensitive immunological assay procedure, which allowed the detection of less than 1 ng (nanogram) of antigen. its selectivity Antigenic differences between the extracts compared were determined against a background of insignificant similarities, whereby the detection arrangement serves as feed-back control of a series of chemical procedures, which were varied to produce tumor fractions with 1) maximal antigenic reactivity with antitumor sera, which were absorbed by normal antigens, and 2) minimal antigenic reactivity with antitumor sera having been absorbed by tumor antigens. For additional control purposes, tumor fractions with the least reactivity with anti-normal sera, which have been absorbed by normal antigens or tumor antigens, were also determined.

Carcinoma tissue and normal tissue in a frozen state are mixed separately with water at a temperature of approx. 0°c and homogenized in such a way that excess frictional heat is not developed, which would lead to inactivation of the applicable CAPA. The temperature of the suspension should not be allowed to rise above approx. 0°C.

The formed cold mixture of liquid homogeneous substance and solid components is mixed with an organic solvent which has the ability to dissolve lipid substances, such as e.g. neutral fat, for example acetone or ethyl ether, at a temperature below approx. 0°C, whereby the solvent treatment aims to remove e.g. lipoid substances and give increased exposure of antigenic groups. The solids are separated from the mixture, suitably by centrifugation, whereby the water and solvent layers are discarded. The temperature should not exceed approx. +4°C.

The obtained solid components are lyophilized and the lyophilized tissue powder is ground, for example in a stainless steel ball mill at a low temperature, preferably below approx. 0°c and appropriately at a temperature of approx. -70°C. The paint resulted in a fine grey-brown powder.

If desired, the formed powder is extracted with a salt solution, such as e.g. a sodium chloride solution, at neutral pfi and low temperature, appropriate approx. 0°C, whereby the overlying liquid is discarded after centrifugation" The resulting sediment can be resuspended in cold water, whereby the sediment is preserved and lyophilized. The resulting powder can be stored for years in closed containers

bottles what approx. 4°C.

Upon extraction of the above polypeptide-containing tumor and normal powders with IL 2 O at alkaline pH, the extracts become cloudy upon addition of NaCl at neutral or slightly alkaline pH. This lack of clarity is largely due to the presence of contaminants of a nucleic acid nature. After centrifugation of the extracts treated with saline, all the activity remains in the clear solution above. This can be subjected to isoelectric precipitation at acidic pH, suitably approx. 4.8, and precipitates formed are dissolved in a phosphate-buffered water solution of pH

about. 7.0.

The solutions obtained by the above method are filtered with a suitable gol of the molecular sieve type in perloform with the ability to fractionate compounds with a molecular weight range ora-comprising 10.10 to 20.10, especially approx. 15.10, whereby the filtration gel is eluted with a buffer of approximately neutral pH and 6 6 6 the fraction 10.10 to 20.10 is taken care of, especially approx. 15.10. The gel or molecular sieve can be chosen from among polyacrylamide gels, fomsaturated dextran gels, agar and agarose gels and equivalent gels or molecular sieves, such as e.g. "Bio-Gel A-50m" (trade name for an agarose gel from Bio-Rad Laboratories, Mttnchen, West Germany; 100-200 mesh, exclusion limit (Daltons) 50.10 c., fractionation interval (Daltons) 10<5> to 50.10 <6>, approximate agarose content 2%) or "Sepharose 2BM (trade name for an agarose gel from Pharmacia Fine Chemicals, Uppsala, Sweden; 60-300/µm, beads, fractionation interval 10 5 to 20.10 6 , approximate agarose content 2%), whereby the gel brought into equilibrium with a suitable buffer solution at pH approx. 7.0 such as Sørensen's buffer (a 1/15m buffer solution based on sodium and potassium phosphates) diluted 1:10 The type of sieve or gel used for this gel filtration is not critical and the only requirement in this regard is that it should have the ability to effect a fractionation of compounds within the molecular weight range indicated above. Another useful gel for the purpose of the invention is<H>Bio-Gel A-150m", (Bio-Rad Laboratories; 100-200 mesh, exclusion limit (Daltons) 150.IO<6>, f fractionation interval 10 6 to * 150.10 6)• Regarding further details regarding gel filtration technique, see H. Determann "Gelchromatography", Springer-Verlag, Berlin, Heidelberg,

New York, 1967. The eluent used for the filtration is a buffer solution with a pH of approx. 7, and in the departing solution the antigenic activity is found in the fraction containing said molecular weight interval1, whereby this fraction is taken care of.

The sedimented active fractions from the above gel filtration are dissolved in a suitable buffer with a pH of approx. 7.0 as e.g. Sørensen's buffer is diluted with 100 mlO, and the resulting solutions are filtered through a column filled with a weak ion exchange gel of the molecular sieve type with weak anion exchange properties, for example a polyacrylamide gel such as "Bio-<5el P-2" (defined below), whereby the column is equilibrated with a similar buffer of pH approx. 7.0. The fraction showing antigenic activity is found in the first fraction of the solution leaving the column and said fraction is taken care of.

The fraction retained from the chromatography on "Bio-Gel P-2" above is subjected to a special procedure developed to further purify CAPA. In principle, this method involves the following steps in A mixture of proteins or conjugated proteins is precipitated by isoelectric adjustment of the pH. The thus obtained gel is mixed with a suitable gel which is chosen with regard to the protein mixture present. The formed mixture of precipitation and gel is layered on a column containing an identical gel with the same isoelectric pH, i.e. the pH that is isoelectric for the proteins to be precipitated in the previous step. The column is then subjected to elution under successively increasing or decreasing pH. In the eluate, care is taken of the fraction(s) containing the desired protein or proteins. Although the method according to the invention is not bound to any particular theory, it is assumed that the separation of the molecular layers in the protein mixture can be explained in the following way

The precipitated proteins are exposed to an ion current which brings one protein after another into solution. The time required for solubilization of the different proteins depends on the ionic strength, pH, temperature and flow rate. Since the distribution coefficient for each dissolved protein is lower than for the ion gradient, the protein moves faster in the gel than in the gradient. As a result, the protein will re-precipitate and become stationary until it is again dissolved by the approaching ion front. This procedure is repeated an infinite number of times, which results in the separation of molecular species that differ only slightly from each other.

"Bio-Gol P2" is a crosslinked polyacrylamide (100-200 mesh) with

a molecular weight exclusion limit of approx. 2000, but this exclusion limit is not critical. Other applicable gels are cross-linked, narrow or • such as, for example. "Sephadex 0-25", which has a molecular weight exclusion limit of approx. 25,000, but this exclusion limit is not critical. These gels are of the molecular sieve type and are normally used in 1 bead form. Any such molecular sieve type gel having molecular weight exclusion limits of at least about 2-3000 can be used satisfactorily. For an overview of gels of the molecular sieve type that are suitable for golf filtration in combination with gradient elution according to the invention, reference is made to Gellotte, Fractionation of Proteins, Peptides and Amino Acids by Gel-filtration, in "New Biochemical Separations", Van Nostrand, London/New York, 1964.

The normal and CAPA fractions obtained from the above chromatography. exposed to active substance elimination at pH approx. 5, whereby the sediment is stored and dissolved in water with a pH of

about. 8.5. The pH of the clear solutions is then brought to an acidic pH, suitable for approx. 5.0, with formic acid, which treatment results in precipitation. Each layer is mixed with a seeding of a similar gel which has been brought into equilibrium with HCOOH-HCOONH^ at a pH of approx. 5.0. Each of these mixtures is then placed on a gel column brought to equilibrium according to the above.

For elution of the fines, a pH gradient is used, in this case with successively decreasing pH. A suitable buffer system is HC00H-HC00NH4 and NH4HC03-NH3. The antigenic activity is found in the outgoing solution within the pH interval from the original pH to approx. 3.

The outgoing fraction that is maintained within the pH interval specified above contains the desired CAPA, which can be maintained by freeze-drying. In order to determine the raw molecular weight for CAPA in the aforementioned out-

fraction, the fraction can be subjected to filtration on a gel column which has been brought into equilibrium with HCOOH-HCOONil^ at a pH of

about. 3.0, whereby the gel is selected from those mentioned above. "Bio-Gel P-30" (trade name for a polyacrylamide gel from Bio-Rad Laboratories, Mflnchen, West Germany; 100-200 mesh, exclusion limit (Daltons) 40,000, fractionation interval (Daltons) 2500-40,000), and "Sephadex G- 75" or "G-50" (trade name for dextran from Pharmacia Fine Chemicals, Uppsala, Sweden" 40-120 and 50-150 µm, fractionation interval 3-70,000 and 1-30,000, respectively) are particularly suitable gels for the performance of this filtering. They eluted

the fractions of CAPA with a spectrophotometric absorption peak wavelength within the range of ca. 229 to ca. 233 nm and a molecular weight of from approx. 20,000 to approx. 27,000 is collected.

CAPA can, after filtration, be recovered from the active fraction by freeze-drying and can be stored for a long time in vacuum, cold and darkness.

The isolated material consists of a polypeptide based on

a single peptide chain, as can be seen by treatment with permauric acid. CAPA exhibits a basic reaction and is soluble in the pH range 1-3.5. The unprotected polypeptide is irreversibly denatured at neutral pH, namely at pH exceeding approx. 4.5. The antigen is hygroscopic, turning yellow and inactivated and sticky when it absorbs moisture.

Purified CAPA contains a fluorescent group that can be activated at a wavelength of approx. 288 nm while emitting light with a wavelength of approx. 350 nm. The fluorescence is proportional to the amount of CAPA and can be used to determine the presence of polypeptide during the isolation procedure.

CAPA exhibits spectrophotometric peak absorption at a wavelength in the interval from approx. 229 to approx. 233 nm. In addition, it shows strong tendencies to aggregation and cannot be filtered on normal filter media used for sterilization. It has been shown that CAPA originates from the cancer cell walls, since antibodies obtained by injecting CAPA into a living animal cause complete lysis or splitting of the cancer cells in vivo when such cells are exposed to the action of such antibodies.

As previously indicated, the polypeptides have a molecular weight in the range of 20,000 to 27,000, especially approx. 24,000. Analyzes show that it does not contain carbohydrates and spectrophotometry shows that CAPA is free of nucleic acids. Analyzes performed with the amino acid analyzer with and without oxidation show agreement with the molecular weight as determined by gel filtration on "Bio-Gel P-30" or its equivalent.

Isolated CAPA can be used in many applications, such as

in the production of monospecific antibodies, for diagnostic purposes, for immunization procedures and as an active ingredient in mixtures useful as immunizing agents.

In connection with the production of antibodies which are monospecific with regard to polypeptide antigens which are linked to cancers, a new method has been developed. As stated above, unprotected CAPA is irreversibly denatured at neutral pH, meaning that injection of an acidic solution of CAPA into a live animal results in denaturation of CAPA as soon as it comes into contact with the neutral body fluids. To & prevent such denaturation, an oil emulsion is prepared in which a water solution of CAPA at a pH in the range from approx. 2 to approx. 3 constitutes the enclosed phase surrounded by the oil phase. When such an oil-emulsion is injected into a living animal, the acidic water solution of CAPA is protected by the surrounding oil phase in contact with the neutral body fluids. In this way, CAPA maintains its activity in vivo and it can be transported to the antibody-producing cells.

One way to maintain the antigenic activity in vitro at neutral pH is that CAPA binds complex with inert protein,

like for example. human or animal albumin. By working in this way, a complex is formed which is soluble at neutral pH while the antigenic activity in vitro is maintained.

With regard to cancer diagnosis, a modified hemagglutination inhibition technique has been developed. This technique makes it possible to determine the presence of CAPA at different stages of the cancer process by examining material of interest, such as e.g. patient sera, tissues, surgical samples, punctures and smear samples. This new modified hemagglutination inhibition technique consists of a method comprising the steps a) preparation of a series of samples of the material to be examined by serial dilutionj b) addition to each of said samples of a predetermined amount of antiserum containing antibodies specific to

consideration of CAPAi

c) adding after incubation to each of said incubated samples a predetermined amount of CAPA carried by a particle source

carrier#

d) comparing the obtained series of treated samples with control samples of decreasing known amounts of CAPA causing inhibition and predetermined amounts of antiserum containing said antibodies and then adding to each of said control samples a predetermined amount of CAPA supported by a similar particulate carrierj and e) determination of the amount of CAPA in the examined material by comparison of said test series and said control samples for

to detect the presence of cancer and its stage of development.

The invention also provides a mixture that can be used as<i>mmuniserin<g>smiddGl which is active in vivo, which mixture contains here defined CAPA and a pharmaceutically acceptable carrier. The term "living animal" is meant in the present description to also include humans.

The invention will now be described further by means of the following non-limiting examples.

Example X

isolation of pure antigen

As it has been shown (Int. Arch. Allergy 36, 191-203

(1969), “Systematic Antigenic Change in Human Carcinoma Tissues

by Hemagglutination Techniques" by B. Bjdrklund) that most cancer tissues contain the antigen according to the present invention, cancer tissues of different types and from different localizations were collected to create a tumor pool. In this case, the pool was made of macroscopically pure, histologically verified, carcinomatous tissue from the following organs (autopsy) in the chest, bronchi, appendix, colon, duodenum, gall bladder, kidney, larynx, liver, lung, oesophagus,

fallopian tubes, pancreas, prostate, rectum, stomach, uterus.

Normal tissue was collected from a number of individuals to obtain a pool of isoantigens, tissue antigens and Individual antigens. Autopsy style trap was used and the tuiuor diagnoses were histologically verified. Furthermore, it was confirmed that said tumor antigen was intact due to immunological cross-activity with new tumor tissue and living tumor cells. Aseptic conditions were maintained where possible.

Carcinomatous and normal tissue from autopsies was freed separately from adjacent tissue and was cut into 2-3 cm cubes, which were washed in 0.9% NaCl at 0°C until they no longer gave off a blood color. The washed cubes were frozen and stored at -24°C.

Still in the frozen state, the tissue cubes were mixed with 10 ml of H2O at 0°C for each gram of frozen tissue and homogenized in a chilled RSfi Ato-Mix homogenizer with rotating blades at "half speed" during three time periods of 1 min. In this context, it should be observed that the temperature of the suspension should be kept at approx. 0°C, preferably somewhat below said temperature.

Pour the cold homogenate into cold 1000 ml polyethylene bottles. 400 ml of suspension and 400 ml of ethyl ether at -20°C ("Aether ad Narcosin pH Nord., cont. diphenylamine 0.002%" from Skånska Bomulla-krutfabriken AB, Dosjebro, Sweden) were mixed in each bottle and shaken at -2°C for 120 minutes in an International PR-2 centrifuge shaker at a rotor speed of 300 rpm. The mixture was centrifuged at 1000 x G at -2°Ci for 5 min. At this point, the lipid layer does not mix with the tissue layer. Ethyl ether and the lipid layer were discarded and a second extraction was performed for 60 min. followed by centrifugation for 5 rain. and removing the ether lipid layer. Finally, centrifugation was performed at 1000 x G at -2°C for 30 minutes and the aqueous layer was removed if it contained any CAPA. Remaining insoluble constituents were retained. Residual ethyl ether was removed by applying vacuum for 5 minutes.

The antigenic material was suspended in 50 ml H.O

0 o C, transferred to 500 ml infusion flasks and shell frozen at -70 o C (dry ice and ethanol).

Lyophilization was carried out in a Texvac, Type IV A, lyophilizer (Textor, Bad {Joden, Taunus, West Germany) at controlled temperature

The lyophilized tissue powder was ground in a stainless steel ball mill ("Cytolator", Lars Ljungberg Company, Stockholm, Sweden) at a temperature of approx. -70 C for 2 hours at 40 orodr/rain. The paint resulted in a fine grey-brown powder. This powder was extracted with pre-cooled ethyl ether at -2°C in a PR-2 shaker at 300 rpm for 60 minutes and centrifuged at 1000 x G at -2°C for 30 minutes. The ether layer was removed and the sediment dried in vacuum. The obtained crude tissue powder (CTP) can be stored for years at 4°C in closed bottles without significant loss of activity. 5 g of CTP was suspended at neutral pH in 500 ml of sodium chloride solution containing sterile ice cubes. The suspension was homogenized in an MSE Ato-Mix at "half speed" for 3 periods of 1 min. The temperature was kept at 0°C. The mixture was kept at 0°C for 30 minutes with slow stirring. Centrifugation was carried out at 1000 x G at 0°C for 40 minutes. Then the above liquid was discarded.

The sediment was resuspended in 500 ml of cold, distilled water and stirred slowly at 0°C for 30 minutes. After centrifugation at 1000 x G at 0°C for 40 minutes, the above solution was discarded. The remaining sediment was suspended in 25 ml of cold, distilled water and shell frozen at -70°C (dry ice and ethanol). The lyophilization resulted in a washed tissue powder (WTP), which can be stored for years in closed bottles at 4°C.

A water extract was made of CAPA and normal WTP wood

pH 9.5 and this extract was concentrated 10 times by isoelectric precipitation at pH 4.8 and the precipitate dissolved in .1/10 of the volume of water at pH 9.5. The obtained concentrate was stabilized by rapid heating to a temperature of 98-100°C, which temperature was maintained for 5-10 minutes. This heat treatment destroys enzymes, which would otherwise inactivate CAPA. The stabilized extract becomes cloudy upon addition of sodium chloride solution at pH 7.5 as a result of the removal of residual contaminants of a nucleic acid nature. To remove such remaining contaminants, the removal was carried out in 8.0 ml of CAPA and normal water extracts separately by adding 0.8 ral of a 10% solution or NaCl. After centrifugation at 27,000 x G for 1 hour, the clear supernatant solutions were subjected to isoelectric precipitation at pH 4.8 (pH buffering with 0.ln ICl or 0.ln HCl). The precipitates were dissolved in 2-3 ml m/150 phosphate buffer, pH 7.

The obtained solutions derived from tumor and normal WTP were filtered with "Bio-Gel A-50ra" (as previously defined) in cooled columns with an inner diameter of 2.5 cm and a length of 138 cm at a flow velocity of 6.3 ml/cm<2>Vhour. The gel was brought into equilibrium with m/150 phosphate buffer with arrow 7.0 with 2% butanol and eluted with the same buffer. For each charge, 4-8 ml of extract containing a total amount of 50-100 mg of protein was used.

The antigenic activity was recovered in the intermediate fraction of the exuding tumor extract. 1.0 ral aliquots of this fraction, totaling 160-180 ml, were diluted 3 times and adjusted to different pH values ranging from 2.0 to 9.0. After 5 minutes at room temperature, the samples were transferred to cuvettes and their light scattering at 500 nm was measured under an angle of 90°. The scattering intensity was plotted against pH and indicated a maximum at pH 4.8, which pH constituted the fraction's isoelectric point. This method is a new technique that has been developed for the purpose of determining the exact and necessary conditions for the purification of CAPA. Conventional techniques for determining the isoelectric point cannot be used due to the peptide's low concentration.

The remaining part of the active intermediate fraction was precipitated at pH 4.8 using 0.ln HCl. Centrifugation at 3800 x G at 0°C for 5 minutes resulted in quantitative yield of activity.

To achieve further purification of CAPA, the sedimented, active me 11 omf r ax ion une from five "Bio-Gel A-50m" columns were dissolved in m/150 fos f r.tbuf solution'with'pl-l 7.0 (Sørensen's buffer in 2% N-butanol) to 5% of the original volume, berm solution com containing 130 mg of protein in 8.5 ml was filtered through a "Dio-Gel P-2" column (as previously defined) with a diameter/height ratio of 1/35 and at a flow rate of 35 ml/cm''/hr. The column had been brought into equilibrium with m/150 f os»f atbuf f is with pli 7.0 (Sørensen's buffer in 2% N-butanol). For each rag protein, a layer volume of 10 ml was allowed.

The first fraction was active and was excluded with elution buffer of pH 7.0 while contaminants were absorbed on the gel at the low ionic strength used. The active substance was precipitated at pli 4.8 with 0.1 µM HCOOH and centrifuged at 3800 x G at 0°C for 5 minutes. The sediment was dissolved in 8.5 rui H^O and pyr. adjusted to 8.5 with 0.lm NH40H. The clear solution was resuspended 3 times with 0.1 µm HCOOH at pH 4.8, washed in the centrifuge at the same arrow and dissolved at pli 8.5. The removal of N-butanol was followed by gas-liquid chromatography. The finished solution was stored in ampoules, shell-frozen and lyophilized. Lye formed product was a dry, almost white powder and this powder was stored at -24°C

in evacuated and closed ampoules.

An identical procedure was applied with a corresponding extract derived from normal tissue and no activity was obtained.

For further purification of the product obtained from the previous chromatographic procedure, a pH gradient elution method was developed. This method basically consists of the following steps: A protein mixture which is to be purified with respect to the active component in them is precipitated by means of isoelectric adjustment of pH. The gel is mixed with a gel, for example "Sephadex G-25" or "Bio-Gel P-2". This mixture was layered on top of a column containing an identical gel with the same isoelectric point. The column is then subjected to pH gradient elution under constant flow rate and temperature. The column is thus eluted with a medium of continuous or stepwise increasing or decreasing pH.

In the present case, gradient elution was performed with 15 mg of lyophilized and salt-free CAPA powder and normal antigen powder that has been purified as described above, in this case at decreasing pH. The powders were dissolved separately in H 2 O, whereby pli was adjusted to 8.5 against 0.ln NH 2 OH. The clear solutions were brought to 5.0 with 0.1 m HCOOH, which resulted in purification. Each pellet was mixed with 10 ml of "Bio-Gel P-2" slurry equilibrated with an aqueous solution made of 0.02 m HCOOH and 0.02 ra HCOONH 2 to achieve a pH of 5.0. Each of these mixtures was placed on top of a 15 ml silicone lined "Bio-Gel P-2" column with an inner diameter of 16 mm and a length of 150 mm which was brought to equilibrium as above. The gel was supported by a small piece of glass wool.

A pH gradient was used to elute the fines. The pH was maintained using a gradient mixer (Ultrogrod, LKB, Sweden).

at approx. 5 for a short period of time for washing the fines, after which the arrow was successively lowered to 2.8. Finally, the pH was raised to 3. The buffer system comprised 0.02m HC0011-HC00KH4 and 0.02m Uli^ aCO^- mL^ and the elution was carried out at room temperature. The flow rate was maintained at 27.2 ml/cm<2>column cross-section/hour.

The fluid leaving the tumor extract was analyzed by means of fluorescence spectrometry (activation at 288 nm, fluorescence emitted at 350 nm). The fraction that was eluted between the initial pH and approx. 3 was collected for further application. After lyophilization and freeze-drying, the products could be stored in closed ampoules after removal of air at -24°C.

For molecular size characterization and to confirm purity, the antigenic material was filtered in the form of solutions of them with "Bio-Gel P-30" (as previously defined) equilibrated with a buffer 0.02m HC00H-HC00NH4»pH 3.0. The pH-eluted CAPA was dissolved in a small amount of the same buffer. The flow rate was 3.25 ml/cm 2 /hour and the fractions collected contained 1.24 ml. The active fractions showed a spectrophotometric absorption maximum at a wavelength of 229-233 nm and a fluorescence of 350 nm upon activation at 288 nm.

In a typical experiment, 10.4 µg of CAPA was dissolved in 3.0 ml of the above buffer on a silicone-treated "Bio-Gel P-30" column (inner diameter 2.54 cm, length 50 cm) and this resulted in complete recycling of both substance and activity. The elution volume was consistent with a molecular weight in the range of 22,000-24,000. These molecular weight values were obtained by comparing the elution volumes from compounds with known molecular weights, such as e.g. ribonuclease and inculin. After freeze-drying, a white, hygroscopic powder was obtained which can be stored in the cold and in the absence of light and oxygen. When analyzing the products, no carbohydrate and no nucleic acids were found. Analyzes with an amino acid analyzer showed agreement with dm molecular weight determined by gol filtration. Analyzes of the amino acids showed the following percentages, which have an accuracy of 1 5%. The calculated molecular weight was 23.2001 2500 (standard deviation).

This amino acid content corresponds to a theoretical nitrogen content of 16.2-17.8%. Determination of total nitrogen according to Dumas points to a value of 17,010.5%. As shown by binding to perauric acid, the polypeptide is based on a single peptide chain. Furthermore, the polypeptide is irreversibly denatured at a pH exceeding approx. 5 if it is not protected by complex formation with albumin.

Example Ia.

In exactly the same way as described under Example I above, the polypeptide is produced from tissue from the human placenta.

Example Ib

Preparation of CAPA albumin complex

An aliquot of the CAPA prepared in example I is dissolved in formic acid (for example 0.05m; pH should be in the range 2-3). It is achieved

a clear solution. Albumin powder or a solution of albumin is added to said solution at the same rate as in the formic acid solution above (200 parts by weight of albumin per 1 part by weight of CAPA), resulting in a clear solution of CAPA and albumin. The pH of the solution is then increased

slowly by adding a bar. ■■? (okcc-mpslvls tn vcjtml-icing of sodium hydroxide or ammonia) until pil becomes approx. 7.5. Dotte results in a clear solution containing CAPA and albumin in the form of a complex.

This solution containing 12 µg CAPA/ml can be used directly in a diagnostic procedure described below or the complex can be isolated in the form of a white powder by freeze-drying. The fulver is stable in the cold (r4°C) for a long period of time.

It has been shown in experiments that maximum utilization of the CAPA activity is achieved at a weight ratio of albumin/CAPA approximately equal to or exceeding 2CO:1.

Example. Ic

Production of stannous acid-treated and labeled red blood cells

Fresh sheep blood (1 volume part) is added to sterile Aisever's solution (1.2 volume parts) (Alsaver's solution is prepared from 250 g glucose, 80 g sodium citrate dihydrate, 42 g NaCl and water to 10 liters, pH is adjusted to 6.1 against 10% citric acid monohydrate) . The mixture is centrifuged and the red blood cells are suspended once more in Alsever's solution and twice in a buffer solution with pH 6.8. Finally, a suspension of the red blood cells is made in the buffer with pH 0.8

1 a concentration of 10<9> cells/ml.

1 part by volume of suspension of red blood cells prepared according to the above is added to 1 volume of buffer solution with pH 6.8 containing approx. lS^ug stannous acid/ml with stirring. The red blood cells thus treated with stannous acid are centrifuged and suspended twice in a buffer with pH 7.5. Finally, the red blood cells are suspended in a buffer with pH 7.5 in a concentration of 10 9coller/ml.

An aliquot amount of the CAPA complex prepared in example Ib above is dissolved in a buffer solution of 7.5 until a concentration of 3 µg CAPA is formed per ml (calculated on pureCAPA).

1 part by volume of the suspension of stannous acid-treated red blood cells

prepared according to the above is added dropwise to 1 volume of CAPA complex solution at 0°C over a period of 10 minutes The labeled blood cells are centrifuged and resuspended in a buffer solution of pH 7.5 containing a stabilizing amount (about 1.2 volumes/volume) of inert human serum to make a suspension containing 1.6 x 10 8 labeled blood cells/ml. These labeled blood cells are used as shown below in the diagnostic method according to the invention.

Example J. I

Production of antibodies'

In view of the fact that CAPA according to the invention is irreversibly denatured by high-level arrow, as previously stated, it was suspected that parenteral injection of the polypeptide would not result in the formation of antibodies. Experiments carried out on rabbits confirmed this, it was found that no antibody formation occurred after injection of polypeptide complexed with albumin into rabbits. All hemagglutination reactions were negative.

In order to enable transfer of the polypeptide in an active state to the antibody-producing cells, it is necessary to maintain the peptide in solution at pli lower than approx. 3 (0.02m HCOOH, pH 2.8) and emulsifying the solution in oil to form extremely small droplets surrounded by a protective oil layer. The injection of the emulsion resulted in the formation of satisfactory amounts of antibodies, which reacted specifically with CAPA in hemagglutination reactions where blood cells were labeled with CAPA.

Preparation of immunization agent

816 mg of pure CAPA prepared from pooled human cancer tumors was dissolved in 1.0 ml of 0.02m HCOOH at pH 2.8. To the resulting solution, 1.0 ml of adjuvant oil (technically produced by Difco Laboratories, Detroit, Michigan, USA" Bacto-Adjuvant, Freund) was added dropwise while simultaneously emulsifying. The emulsification was carried out according to Freund by means of an injection syringe, whereby the mixture was drawn up and down the syringe to form a uniform, white emulsion of about the consistency of whipped cream. A drop of said emulsion was tested with ice water to confirm that it floated separately and remained intact. The formed, semi-liquid emulsion, among other things,

, used for subcutaneous and intramuscular injections in rabbits.

Immunization of five dogs

In the first injection, so-called complete adjuvant was used and in the subsequent injections, so-called incomplete adjuvant. After initial opening of blood from the ear of the five rabbits for determination of 0~v?j;dier, a total of 408 micrograms of emulsified CAPA at pH 2.8 was injected subcutaneously on both the right and left side. At ten-day intervals, a further three injections were given, the first intramuscularly in both hind legs and then the second and third subcutaneously on both fronts and both hindquarters respectively. Valent of injection oteders -was made to utilize the access points for-the reglanalo lymphokj ertlenc. In total, each rabbit received 1.6 mgCAPA in the form of an emulsion. 14 and 24 days after the last injection, test samples of blood were taken, which were subjected to hernagglutination analysis with regard to the presence of specific antibodies against CAPA. Two days after the last sample, a maximum amount of blood was withdrawn, which was stored in the form of serum which was sterile filtered and transferred in small portions to sterile tubes. These tubes could be stored refrigerated and retained their specificity.

Example III

Diagnostic procedure

a) Simple blind experiment.

Blood samples were obtained from the Central Hospital in Eskilstuna,

Sweden, of which 42 were from patients at the infectious disease clinic;

one from surgical clinic; two from radiotherapy; 10 from newly married mothers;

10 from umbilical cord blood and 10 from pregnant women in the first trimester and

6 in the II trimester. Furthermore, blood was received from 20 10-year-olds

from Slottsskolan and 10 samples from elderly healthy people from Dr. Lundstrom's private clinic in Eskilstuna, Sweden. From the surgical and medical clinic at Ersta Hospital in Stockholm, Sweden, samples were received from 44 patients, from Akeshov's hospital samples were received from 45 patients and from the surgical clinic at Karolinska Hospital in Stockholm, Sweden, blood samples were received from 72 patients who were admitted for possible surgery. The Blood Donor Center at Karolinska Hospital in Stockholm, Sweden obtained 118 samples from donors aged 20-67 years, mean age 37.2 years (SD- 11.3 years), and from the Hospital's Kliniska Central laboratory, sera were received from 29 patients in wood care

, Radiumhemmet's general departments and from 12 patients at its gynecological departments.

The total number of blood samples amounted to 431. For the 111 cases from Eskilstuna, a clinical follow-up was carried out and a medical record was drawn up, apart from the obviously healthy individuals. As far as the Stockholm cases are concerned, detailed medical records were studied for 101 cases. In the remaining cases, there were clinical diagnoses other than malignant and there was no suspicion of malignant disease.

Venous blood was transferred to Wassermann tubes without addition

and sent to the laboratory, in certain cases after removing

clot and blood cells using centrifugation.

The blood analysis was based on the indication of i tarves r of CAP7 using a modified hemagglutination inhibition technique (micro-method). In principle:» the following procedure was used: 25 µl patient serum, which had been absorbed with red sheep blood cells, was titrated in 8-step 2 dilution in Linbro IS-MRC-26 dilution plater, after which 25 µl specific antiserum in limit- dilution (approximately 1:2000) was added to each well. After incubation at 22°C for 10 minutes, 100 ml of the suspension of approx. 6-8 million red sheep blood cells which were tin-acid treated and labeled with isolated CAPA bound to human albumin, as shown in Example 1c above (about 2-4/Ug per 10"' blood cells). Other particle formigo b-Drero, such as latex, bentonite and collodiura, can be used instead of red blood cells. The reaction was read using an inclined mirror and a standard series after 4-24 hours of incubation at 1-2°c For control purposes, it was used" 1. Blood cells labeled with CAPA plus antiserum; 2. Blood cells labeled with CAPA without antiserum; 3. Patient serum plus blood cells labeled with human aibumin; 4. Patient serum and untreated blood cells; 5. CAPA-labeled blood cells and antibodies in a series where inhibition occurs with stepwise decreasing, known amounts CAPA.

The diluents used contained pooled (ie, pooled from many individuals), inactivated, neutral human serum for stabilization of the blood cells.

CAPA reagent was prepared from pooled human carcinoma tissue and purified as described above by gel filtration, pH gradient elution, filtration through "Bio-Gel P-30" and complexation with albumin.

As antiserum, horse anti-HjLa from November 19, 1962, absorbed with normal tissue and pooled human serum was used. The immunization was carried out with washed fragments of HeLa cells grown in Eagle's medium containing 205b inactivated human serum in flat glass bottles. The cells had been harvested with EDTA, centrifuged and washed 3 times with water.

In the presence of CAPA in patient serum, the horse antibodies were neutralized, which meant that the agglutination did not occur when the polypeptide-labeled blood cells were later added. Reading was done according to a rising scale from 0-6, where 0 meant absence of inhibition and 6 indicates m?V«irnjvl inhibition cv the agglutination, flelrsj onon between r$k al of words? i'?, r 07 concentrate on CAPA/ml sarwr. appears in Table 1, which is obtained by calibrating dilutions of predetermined amounts of GAPA in neutral serum.

The readings were carried out without knowledge of the patients' state of health. The material that was the subject of investigation has been grouped, as shown in table 2 below.

It appears from the results that the highest scale values have been achieved with the groups that include "primary cancer with metastases" (regional) and "widespread cancer" such as the group "newborns"

(umbilical cord biod).

Low Bkaia values have been obtained for blood donors. An intermediate position is occupied by the group "primary cancer, untreated" and the group "cancer, not radically treated". Values close to those of blood donors are found in the group "cancers, radically removed".

Mean values regarding CAPA in all groups have been compared in pairs and are shown in table 3 below.

a) Significance tests of differences were carried out using jji approximately normally distributed variable, z defined as: c) This group includes 11 positive tests, of which 8 apply to suspected but not confirmed cancers.

It is obvious from the table above that with regard to "primary cancer" with metastases" as well as "widespread cancer" there are significantly higher mean values than for the other groups with the exception of newborns (see also table 2).

For the groups "canoes, not radically b<'treated" and "priacer cancers, untreated" the mean values are significantly higher in relation to all groups except the two former and to "malignant disease, other than cancer", against which the difference is significant ( 0.01 P . 0.05).

Groups 5-9 do not show significant differences among themselves, apart from the significant difference between 7 and 9. This difference can be explained by the fact that true, hidden cancer cases can occur among the cases showing scale values between 1 and 4.

The positive finding in the umbilical cord biopsy deserves some attention. To determine whether CAPA originates from the placenta was. dot ekntrahort pieces of such tissue. in V; n'ye,; r of 300 mg frozen placentov.-sv per ml' buf rot physiological r-iCi--?.r'.3ru<*>.u'j v :å pH 7.5 was homogenized at 0°C and centrifuged at 1000 :< G for 30 minutes at 0°c i conically elongated tubes (according to Markham). Then the above solutions were tested for CAPA by i) using the hemagglutination technique described above. In order to rule out the possibility that inhibition reactions are obtained if they have been caused by non-specific inactivation of now blood cells i-ag)plied CAPA, the said blood cells were shaken after each measurement and a standard amount of antibodies was added which are specific against CAPA. No antiganin activation could be observed in any of the cases. An extract of blo was used as a control material

■antigen powder prepared from human cancer tissue of different origin and from different local storage organs. The results vxrvv..->t of 26 placentas from the same number of individuals all without exception contained significant amounts of CAPA. The mean value (geometric) for the 26 placentas was 297 l 8 micrograms per g wet tissue (0.03%).

Since it is obvious from the above investigation that CAPA according to the present invention is synthesized in the placenta and since the genes contained in the placenta must also be included in individuals originating from the same egg cell, it follows that the facility or ability to produce CAPA is a normal phenomenon. Normally, this facility is inhibited since normal cells do not contain CAPA in measurable amounts. In cancer cells, on the other hand, there has been an activation of the plant characterized by a noticeable presence of CAPA inside and around the cells. Don antigenic and in functional respects the functional similarity between trophoblaatcoli in placenta and cancer coller and < unlike ho ton musl lom the individuals' other coils seem to correspond to the d!:n rafluction that k a uc • :u* the disease c or an expression of a changed control of normally occurring facilities.

A statistical analysis of the experimental results shows that there is a highly significant correlation between the presence of a canoe diagnosis and elevated CAPA concentration (P <.0.001). The same applies to the relationship between the size of the tumor mass and the elevated CAPA concentration within the same age group (P <0.001). The elevated concentration of CAPA has been observed in connection with many different localizations of cancers. Regardless of type and location, cancer tumors have been shown to

contain CAPA, whereby said CAPA appears to be common to all known types of cancer, i.e. malignant tumors of epithelial origin.

h) l.obb :lt blind experiment

To further confirm the correctness ri v ov.rnst5.--nd i

conclusions, a double blind experiment was carried out under the most rigorous conditions. Coded blood samples were received from the various departments at the Central Hospital in Kskil«tuna, Sweden. In this case, dut was carried out quantitatively..; measurements of the CAPA level in patients' pain".

The results of the double-blind search, as well as the results from the above single-blind trial, are compiled in

tables 4-8 below. The aforementioned tables show:

Table 4 the total number of examined individuals and the number of wt individuals who had a cancer diagnosis;

Table, 5 the number of individuals of the different groups as well as their age and distribution;

Table 6 the presence of CAPA in sera from all examined patients;

Table 7 shows the percentages of individuals who had a CAPA concentration of 0.15 µg/ml serum, excluding groups 9, 10 and 12 in Table 5; and

Table 8 the presence of CAPA 1 in relation to tumor locations for groups 1-3 in table 5. Site numbers refer to "Cancer Incidence in Sweden 1968".

ISn exact analysis of the in that ..' doubtbl indfoxsøk0£>p~reached data shows again the highly "significant ik-p.te horr -l;-' in ion :n in .v 11 about the presence of kanccrdiagno3e and elevated CAPA -conservation (F ✓ 0.001), This double-blind trial also confirms the relationship between the size of the tumor mass in question and the elevated c7vPfi correlation within the same age group (P 0.001). D-1 fore<:>yodo s The jruri/i level of CAPA has been found in agreement with approx. in the new experiences and their practical applicability. In the introductory part of the present bi-r.kr.ivelpo, it is stated that the antigen that was invented in US patent no. 3,663,664 is clearly different from the antigen according to the present invention , foc ■ : show; the difference between d--t known-* antigen ot and antig-m st according to the invention is given below a detailed compilation of certain specific fo differ both with regard to the method for antigen isolation and with regard to the properties of the antigen.

In summary, below are brief definitions of the peptide identification and diagnostic methods according to the present invention.

Definition of CAPA

HeLa cells (originating from a cane pea in the year 195?) are grown in vitro. These cells are used as an antigen source. The harvested cells are injected into the host, which results in the formation of f-t specific antibody .«."if used for the identification of the antigen in tumors, serum, placenta, etc. Let the latter antigen must ba and have an identical, immunological r erection ons~F-.it e (compared to Hej.eO-antigen) , but can naturally vcr-re nnvl cerledes in nnus.^he>v-spender when it is ni*Creating in natural tilr:.tand.

po f j. niai on,, fly., diagnostic, f r enig aue/ small two

Antigen from He.éa~oello cultures in vitro auvendas for the formation of antibody in horses. After observation of undesirable antibodies, the cough serum is used for agglutination of red blood cells labeled with antigen taken from tumors (or placenta or c 1e dy rien ing v >: of Hei.--, or HF.p-2 oll ;-r Detroit-0 ). Lc-tto t. indicator system<g>t. If an unknown sample contains antigenic activity, it thus affects the antibody and inhibits agglutination.

Uonslng .~y_ to. tumor kn ytted; ^nt^the enemy

The floating spoon with greens illustrating the aimindeiiye ren.sn.ings procedure.

Claims (27)

1. Cancer-associated polypeptide antigen (CAPA), characterized in that it has a molecular weight of 23,200-2,500 (SD) and with a spectrophotometric peak absorption at a wavelength within the interval from 229 to 233 nm, which polypeptide is based on a single polypeptide chain as shown by treatment with permauric acid, and, if not protected by complexation with albumin, denatures irreversibly at pH above 5. 2. CAPA according to claim 1, characterized in that the polypeptide contains the following amino acids with the following mole percentage: 3. CAPA according to claim 1, characterized in that it contains a fluorescent group that emits fluorescent light at 350 nm upon activation at a wavelength of 288 nm. 4. Method for isolating a polypeptide antigen that is linked to cancer (CAPA) according to claim 1, characterized in that 1 a) CAPA-containing material is homogenized in a liquid at a temperature not exceeding b°C, b) solid components from the mixture of liquid homogenate and solid components originating from step a) are separated, c) said solid components are extracted with an alkaline water solution, d) the extract from step c) is filtered on a filtration gel of the molecular sieve type in bead form with the ability to fractionate impurities with a molecular weight range comprising 10.10 g to 20.10 6 , in particular about 15.10 6 , the gel is eluted and the 10.10 6 to 20.10 6 fraction, in particular the fraction of about. 15.10 6 stored, e) the fraction from step d) is subjected to chromatography on a weak ion exchanger for absorption on this of contaminants, and the first fraction leaving said ion exchange is retained, f) said first fraction is subjected to isoelectric refinement, g) formed precipitate is mixed with a gel of the molecular sieve type and said gel is subjected to pH gradient elution and h) during lowering of the pH, the fraction is stored down to pH 3 containing CAPA with a spectrophotometric absorption peak wavelength of 229-233 nm and a molecular weight within the interval 20,000 - 27,000. 5. Method according to claim 4, characterized in that the fraction obtained from the pH gradient elution is subjected to gel filtration on a gel column, in which the eluted fractions of antigen have a spectrophotometric absorption peak wavelength at 229-233 nm and a molecular weight within the interval, 20,000 - 27,000, stored. 6. Method according to claim 4, characterized in that the homogenate from step a) is subjected to treatment with an organic solvent in the cold before step b). 7. Method according to claim 6, characterized in that the solvent used is ethyl ether. 8. Method according to claim 4, characterized in that the homogenization is carried out in water at a temperature that does not exceed 0 °C. 1 9. Method according to claim 4, characterized in that the solid components obtained from step b) are lyophilized and then ground in a steel ball mill in the cold. 10. Method according to claim 9, characterized in that the needling takes place at a low temperature. 11. Method according to claim 9. characterized in that the painting takes place at a temperature below the freezing point of the treated material. 12. Method according to claim 9, characterized in that the painting takes place at a temperature of -70°C. 13. Method according to claim 4, characterized in that the filtration during step d) is carried out on polyacrylamide gels, cross-linked dextran gels, agar and agarose gels, the gel being brought into equilibrium with phosphate buffer at pH 7. 14. Method according to claim 4, characterized in that the chromatography during step e) is carried out in a column of a weak ion exchanger at neutral pH. 15. Method according to claim 14, characterized in that the chromatography is performed on a weak ion exchange polyacrylamide gel which is in the form of a column at neutral pH. 16. Method according to claim 4, characterized in that during step g) a gel selected from among polyacrylamide gels, cross-linked dextran gels, aga and agarose gels is used. 17. Method according to claim 5, characterized in that said gel filtration is carried out on a polyacrylamide gel or a cross-linked dextran. 18. Method according to claim 4, characterized in that the water extract from step c) is subjected to rapid heating to a temperature within the range of 95 to 100°C to destroy enzymes. 19. Method according to claim 18, characterized in that said water extract is quickly heated to a temperature within the interval 98-10 O°C and held at said temperature for 5-10 minutes. 20. Method for producing CAPA according to claim 4, in characterized by that antigen-containing material is homogenized in a frozen state in water at a temperature not exceeding 0°C, formed homogenate is subjected to treatment with a solvent selected from ethyl ether and acetone, solid components are separated from the obtained mixture of liquid homogenate and solid components, said obtained solid components are lyophilized and ground in a steel ball mill at a temperature below the freezing point of the treated material, solids formed are extracted with an alkaline water solution and after centrifugation the overlying liquid layer is subjected to rapid heating to a temperature within the range of 95-100°C for a period of 5 - 10 minutes to destroy enzymes, the heated overlying layer is treated with an aqueous solution of sodium chloride for precipitation of impurities and the above liquid is stored, the liquid obtained is filtered on a cold column of a molecular sieve-type gel in bead form with a fractionation interval of 10,000 to at least 20,10 and equilibrated with phosphate buffer at pH 7.0 and the active fraction of the eluate is kept, said active fraction is subjected to chromatography on a column of a weak ion exchange gel of the molecular sieve type equilibrated with phosphate buffer of pH 7.0, and the first fraction of the liquid leaving the column is retained, and said first fraction is subjected to isoelectric precipitation at pH 5.0 and the precipitate formed is mixed with a molecular sieve type gel having a molecular weight exclusion limit of at least 2000 equilibrated with HCOO H-HCOO NH^ at pH 5, the resulting mixture is applied to a column containing an identical gel brought to equilibrium in the same way, the column is pH-gradient eluted and, while lowering the pH, the fraction eluted down to pH 3 containing CAPA with a spectrophotometric absorption peak wavelength at 229-233 nm and a molecular weight of 23,200 2,500 ( SD). 21. Preparation usable as an immunization agent, characterized in that it comprises as active ingredient CAPA according to claim 1 in combination with a pharmaceutically acceptable carrier. 22. Preparation according to claim 21, characterized in that it comprises an oil emulsion in which a water solution with a pH lower than 3 containing CAPA forms an enclosed phase which is protected by the surrounding oil phase. 23. Preparation according to claim 21, characterized in that the oil emulsion is based on Freund's adjuvant. 24. Preparation according to claim 21, characterized in that it contains CAPA and a stabilizing amount of inert protein, preferably albumin in mixture with it. 25. Preparation according to claim 24, characterized in that the ratio between albumin and C APA is at least 200:1 calculated by weight. 26. Process for the production of CAPA-albumin preparation according to claim 24, characterized in that CAPA and albumin are mixed in an acidic solution and that the pH of the solution is raised to achieve a synergy between the components. 27. Method according to claim 26, characterized in that the preparation is lyophilized.