DE2366609C2 - - Google Patents

Description

The presence of antigens associated with Dick's andenocarcinoma intestine and the digestive system are connected in J. expt. Med., 121 (1965), pages 439 to 462 and J. expt. Med. 122 (1965), pages 467 to 487. In these Be is referenced to earlier work by B. Björklund, the existence of human, with cancer linked antigen show which is in the larger part of all known malignant tumors with epithelial origin common (see Internat. Arch. Allergy and Appl. Immunol. 1966, 8, page 179 and boarding school. Arch. Allergy and Appl. Immunol. 1958, 12, page 241). In US-PS 36 63 684 is a karzi nomembryonic antigen, the so-called "CEA- Activity "shows. However, this antigen is completely ver differed from the antigen used in the invention, what is shown in detail below.

In boarding school. Arch. Allergy 36, pages 191 to 203 (1969) it is reported that in human cancer cells and He-La- Cell antigens have been identified. Isolation of the Antigens did not, however, and on page 202 is out expressly said that the nature of the tumor antigen is impenetrable be visible. About the possibility of determining An make a statement about the developmental stages of cancer none of the literature cites anything to do to take.

From E. A. Kabat, Introduction to Immunochemistry and Immunolo gie, pages 28/29, Springer-Verlag (1971) is general known with the help of in oil, especially in Freund's Adju vans to immunize emulsified antigens. Especially at the production of antigen related to cancer specific antibodies is the emulsification of the antigen as dry powder or solution in Freund's adjuvant from "J.  Exp. Med. "121, page 441 (1965) and" Int. Arch. Allergy " 36, page 194 (1968). For the production of against This procedure suggested CAPA monospecific antibodies but fail.

The object underlying the invention now existed rin, a process used to make against cancer linked antigen (CAPA) monospecific antibodies to get.

This object is achieved with the method according to the invention solves, in which in stage b) that in stage a) prepared antigen with oil, especially with Freund's Adju vans, processed into an oil emulsion and then live it injected into the animals, and which is characterized by that in step a) an aqueous solution of the antigen with a pH of less than 3.

Preferably, the aqueous solution in step a) below Use a weak acid, preferably ants acid, for acidifying.

A material that has CAPA activity can be obtained by homogenizing malignant tissue from autopsies and collects carcinoma tissues of various types and locations.  

A raw material that can be used instead is tissue from human placenta or cultures of malignant cells in vitro (for the details of such cultures, see B. Björklund, V. Björklund, and I. Hdlöf, J. Nat. Can cer Inst., 26, pages 533 to 545, 1961 and "Antigenicity of Pooled Human Malignant and Normal Tissues by Cytoimmuno logical technique. III. Distribution of tumor antigen "). Normal tissues become parallel to the malignant tissues also collected. Isolation of a pure antigen can be done as follows:

Cancer tissues and normal tissues are frozen mixed with water at a temperature of about 0 ° C and homogenized in such a way that no excess Rei exercise heat is generated, which inactivates the subject antigen. The temperature of the Sus pension should not be read above 0 ° C sen.

The resulting cold mixture of liquid homogenate and solids with an organic solvent with the ability to dissolve lipids like neutral fats such as acetone or ethyl ether, at a temperature treated at about 0 ° C, which is a removal among other things of lipoid substances for the purpose and leads to increased exposure of antigenic groups. The solids of the mixture, expediently by centrifugation, separated, and the aqueous layer and the solvent layer are discarded. The temperature should be around 4 ° C do not exceed.

The solids obtained are lyophilized, and the lyo philized tissue powder is, for example, in a Ku Gel mill made of stainless steel, at a low tempera ground, preferably below about 0 ° C and expedient ßig at a temperature below about -70 ° C. Grinding leads to a fine gray-brown powder.  

If desired, the resulting powder is mixed with a Saline solution, such as with a saline solution, at a neutral pH at low temperature, purpose extracted moderately at about 0 ° C, the according to the centri the top layer is discarded. The result sediment can be resuspended in cold water whereupon the sediment is collected and lyophilized. The resulting powder can remain closed for years Flanges can be stored at around 4 ° C.

After extracting the powder with water at an al The extracts are calic pH when adding NaCl at a neutral or slightly alkaline pH. This cloudiness is mainly due to the presence of impurities of nucleic acid character. After this Centrifuge the saline-treated extracts all activity remains in the clear liquid. The It can be isoelectric at an acidic pH, expediently at about 4.8, undergo precipitation, and the resul Turing precipitation is in an aqueous with Phos phat buffered solution with a pH of about 7.0 solves.

The solutions resulting from the above process are filtered with a suitable molecular sieve gel in pearl form, which allows a fractionation of compounds with a molecular weight range of 10 × 10 ⁶ to 20 × 10 ⁶ , in particular about 15 × 10 ⁶ , the molecular sieve with a buffer of approximately neutral pH is eluted and the molecular weight fraction of 10 × 10 ⁶ to 20 × 10 ⁶ , in particular of approximately 15 × 10 ⁶ , is obtained. The molecular sieve can be a polyacrylamide gel, cross-linked dextran gel, agar and agarose gel or equivalent gel. The gel with a suitable buffer solution at a pH of about 7.0, such as Sörensen-buffer _^(1/15 molar buffer solution, based on the sodium and potassium phosphates), diluted 1: 10, äquili-calibrated. With regard to further details of the gel filtration method, reference is made to H. Determann "Gel Chromatography", Springer-Verlag, Berlin-Heidelberg-New York (1967). The eluent used for the filtration is a buffer solution with a pH of about 7, and in the outlet there is antigen activity in the fraction containing this molecular weight range. This faction is won.

The active fractions from the above gel filtration in a suitable buffer with a pH of about 7.0, like a Sörensen buffer diluted to 1:10, dissolved, and the resulting solutions are fil through a column triert with a weak ion exchange molecular sieve gel with weak ion exchange properties, as with for example, a polyacrylamide gel is filled, the Column with a similar buffer with a pH of about 7.0 is equilibrated. Fraction showing antigen activity is the first fraction of the solution that leaves the column, and this fraction is won.

This fraction is further purified as follows: a mixture of proteins or conjugated proteins is by iso electrical adjustment of the pH value failed. The one there Precipitation obtained is ver with a suitable gel mixes. The resulting mixture of precipitation and gel is applied as a layer on a column that has an identi gel of the same pH value, which is iso for the proteins is electrical and this in the previous stage falls, contains. The column will then gradually become less Elution increase or decrease in pH. By doing Eluate are the fraction or fractions obtained those that have the desired protein or protein included.

Crosslinked polyacrylamide with gels that can be used for this purpose a molecular weight cutoff of about 2000 or cross-linked dextran gels, for example a molecular gene have a weight exclusion limit of approximately 25,000. These gels are molecular sieves and are usually used in pearl form  turns. Any such molecular sieve gel with molecular weight weight exclusion limits of at least about 2000 to 3000 can be used.

The normal fraction and the CAPA fraction obtained in the above chromatography are subjected to precipitation of the active substance at a pH of about 5, whereby the sediment is recovered and in water with a pH of about 8 , 5 is resolved. The clear solutions are then acidified with formic acid, conveniently to a pH of about 5.0, and this treatment leads to precipitation. Each precipitate is mixed with a slurry of one of the above gels, which has been equilibrated to about pH 5.0 with HCOOH-HCOONH ₄ . Each of these mixtures is applied to a gel column equilibrated as above.

A pH gradient is used to elute the precipitates, in this case with a gradually decreasing pH. A suitable buffer system is HCOOH / HCOONH ₄ and NH ₄ HCO₃ / NH ₃ . The antigen activity is found in the outlet in the pH range from the initial pH to about pH 3.

The leakage fraction obtained in the above-mentioned pH range tion contains the desired CAPA (Po associated with cancer lypeptide antigen), which are obtained by freeze-drying can.

The CAPA can be removed from the active fraction after filtration obtained by freeze drying and in vacuum for a long time, be stored in the cold and in the dark.

The isolated antigen consists of a polypeptide on the The basis of a single peptide chain, as by treatment with performic acid. The CAPA shows basic Reaction and is at a pH in the range of about 1 up to 3.5 soluble. The unprotected polypeptide denatures irreversible at neutral pH values, namely at pH values above 5. The antigen is hygroscopic and turns yellow,  inactivated and greasy when it absorbs moisture.

The purified CAPA contains a fluorescent group, that when activated at a wavelength of about 288 nm fluorescent light with a wavelength of about 350 nm emitted.

The CAPA shows a spectrophotometric peak absorption at a wavelength in the range of about 229 to about 233 nm.

The CAPA also shows a strong tendency to aggregate and is on normal filter media for sterilization used, not filterable. It has been proven that the CAPA comes from the cancer cell walls because antibodies, by injecting the CAPA into a live animal body were formed, a complete decomposition of Krebszel cause len in vivo when such cells are under the influence of sol antibodies are exposed.

The CAPA has a molecular weight in the range of 20,000 to 27,000, more specifically of 23,200 ± 2,500, especially of about 24,000. Analyzes show that it contains no carbohydrate, and spectrophotometry shows that the CAPA is free of Nuc is linseed acid.

As stated above, the unprotected CAPA denatures irreversible at neutral pH values, which means that a Injection of an acidic solution of the CAPA into a living Carcasses for immediate denaturation of the CAPA leads when this comes into contact with the neutral body liquids is coming. To prevent such denaturation an oil emulsion is prepared, in which an aqueous CAPA solution with a pH of less than 3 makes up the disperse phase, which is different from the oil phase give is. When injecting such an oil emulsion into one live carcasses becomes the acidic aqueous solution of the CAPA protected by the surrounding oil phase when in contact comes with the neutral body fluids. In this way  the CAPA retains its activity in vivo and can add to the antibody-producing cells.

example A. Isolation of pure antigen

Cancer tissues of various types and types len was collected. It consisted of macroscopically pure, histologically confirmed cancer tissues from the following Or ganen (autopsy): breast, bronchi, appendix, colon, Duodenum, gallbladder, kidney, larynx, liver, lungs, Esophagus, ovary, pancreas, prostate, rectum, stomach, Uterus.

Normal tissues were collected from a number of people melts to a cluster of isoantigens, tissue antigens and get single antigens.

The cancerous tissues and normal tissues were from neighbor tissue freed and cut into 2 to 3 cm long cubes ten that were washed in 0.9% NaCl at 0 ° C until they stopped giving blood. The washed cubes were frozen and stored at -24 ° C.  

While still frozen, the tissue cubes were mixed with 10 ml H ₂ O (0 ° C.) per gram of frozen tissue and homogenized in a cooled mixed homogenizer with rotating knives at half speed for three periods of 1 min each. Care was taken to ensure that the temperature of the suspension was kept at about 0 ° C., preferably somewhat below this temperature.

The cold homogenized mixture was poured into cold 1000 ml Po poured bottles of ethylene. 400 ml suspension and 400 ml Ethyl ether of -20 ° C with a content of 0.002% diphenyl amine were mixed together in each bottle and 120 min at -2 ° C in a shaking centrifuge with a motor speed of 300 rpm moved. The mixture was mixed with Centrifuged 1000 G at -2 ° C for 5 min. During this Stage, the lipid layer did not mix with the tissue layer. The ethyl ether and lipid were discarded, and a second extraction was carried out for 60 min, whereupon centrifuged for 5 min and the ether lipid layer was removed. Finally, at 1000 G Centrifuged at -2 ° C for 30 min, and the water layer was ent even though it contained some CAPA. The remaining un soluble components were taken, the remaining ethyl ether was removed by applying vacuum for 5 min.

The antigenic material was suspended in 50 ml H ₂ O at 0 ° C, transferred to 500 ml infusion bottles and frozen at -70 ° C (dry ice and ethanol).

It was placed in a lyophilizer at controlled tempera lyophilized.

The lyophilized tissue powder was in a ball mill made of stainless steel at a temperature of around -70 ° C for 2 h at 40 rpm to a fine, gray-brown powder ground. This powder was precooled with ethyl ether of -2 ° C in a shaker at 300 rpm during Extracted for 60 min with 1000 G at -2 ° C for 30 min  centrifuged. The ether layer was removed and the Sedi ment dried in vacuum.

5 g of the resulting raw tissue powder were neutral pH suspended in 500 ml saline, the sterile Contained ice cubes. The suspension was in a mixer at half speed for three periods of 1 min homo embarrassed. The temperature was kept at 0 ° C. The Ge The mixture was kept at 0 ° C. for 30 min with slow stirring. It was centrifuged at 1000 G at 0 ° C for 40 min. The above standing liquid was poured away.

The sediment was again distilled in 500 ml cold Suspended water and slowly ge at 0 ° C for 30 min stirs. After centrifuging at 1000 G at 0 ° C for The above floating layer was discarded for 40 minutes. The remaining sediment was cold distilled in 25 ml Water suspended and at -70 ° C (dry ice and ethanol) frozen. Lyophilization resulted in a washed tissue powder.

Aqueous extracts of CAPA and normal tissue washed powder having a pH value of 9.5 were prepared and times concentrated by isoelectric precipitation at pH 4.8 and dissolution of the precipitate in ¹ / _10th the volume of water of pH 9.5 10th The resulting concentrate was stabilized by rapid heating to a temperature of 98 to 100 ° C for 5 to 10 minutes. This heat treatment destroys enzymes that would otherwise inactivate the CAPA. The stabilized extract became cloudy with the addition of saline solution of pH 7.5 due to the precipitation of residual impurities of nucleic acid character. In order to precipitate such residual impurities, 8.0 ml of CAPA and normal aqueous extracts were precipitated separately by adding 0.8 ml of a 10% solution of NaCl. After centrifuging at 27,000 G at 0 ° C for 1 h, the clear, above-floating liquid was subjected to isoelectric precipitation at pH 4.8 (pH adjustment with 0.1 N HCl or 0.1 N HCOOH). The precipitates were dissolved in 2 to 3 ml m / 150 phosphate buffer, pH 7.0.

The resulting solutions, which originated from tumor and normal tissue powder, were cross-linked with the polyacrylamide defined above in cooled columns with an inner diameter of 2.50 cm and a length of 138 cm with a flow rate of 6.3 ml / cm ² / h filtered. The gel was equilibrated with m / 150 pH 7.0 phosphate buffer with 2% n-butanol and eluted with the same buffer. 4 to 8 ml of extract totaling 50 to 100 mg of protein were used in each run.

The antigen activity was found in the middle fraction of the Discharge of the tumor extract. 1.0 ml portions of this frak tion, a total of 160 to 180 ml, are diluted 3 times and to various pH values in the range from 2.0 to 9.0 poses. After 5 min at room temperature, the samples were opened Cuvettes transferred and their light scatter at 500 nm measured at an angle of 90 °. The scatter intensity plotted against the pH value resulted in a maximum pH 4.8, this pH being the isoelectric point of the Fraction is.

The remaining part of the active middle fraction was at pH 4.8 precipitated with the aid of 0.1 N HCl. Centrifuge with 3800 G at 0 ° C for 5 min resulted in a quantitative Activity yield.

In order to effect a further purification of the CAPA, the sedimented active middle fractions from five polyacryl amide columns were diluted to 20 times the original outlet volume with m / 150 phosphate buffer solution of pH 7.0 (soensen buffer in 2% n-butanol) . This solution, which contained 130 mg of protein in 8.5 ml, was passed through a polyacrylamide column (as defined above) with a ratio of diameter to height of 1/35 and with a flow rate of 35 ml / cm ² / h filtered. The column had been equilibrated with pH 7.0 m / 150 phosphate buffer (Sörensen buffer in 2% n-butanol). A layer volume of 10 ml was permitted for each milligram of protein.

The first fraction was active and was using the elution buffer separated from pH 7.0 while impurities on the gel was absorbed at the low ionic strength applied the.

The active substance was precipitated at pH 4.8 with 0.1 M HCOOH and centrifuged at 3800 G for 5 min. The sediment was dissolved in 8.5 ml H ₂ O and the pH was adjusted to 8.5 with 0.1 m NH ₄ OH. The clear solution was reprecipitated 3 times with 0.1 M HCOOH at pH 4.8, washed in the centrifuge at the same pH and dissolved at pH 8.5. The disappearance of n-butanol was followed by gas-liquid chromatography. The finished solution was stored in ampoules, jacket frozen and lyophilized. The resulting product was a dry, almost white powder, and this powder was stored at -24 ° C in the evacuated and sealed ampoules.

An identical procedure was followed with a corresponding one Extract that came from normal tissues and was not Activity found.

For further cleaning of the Chromatogra above The product obtained by the method was an elution method developed with pH gradient. This procedure consists of Principle of the following stages: That regarding its active The protein mixture to be purified is separated by an iso electrical adjustment of the pH value selected. The out precipitation is mixed with a suitable oil. This Ge Mix is applied as a layer to a column, the one identical gel with the same isoelectric pH contains. The column is then eluted with a pH gradient under constant flow rate and temperature below pulled. So the column is continuous with a medium  or gradually increasing or decreasing pH.

In the present case, pH gradient elution was performed with 15 mg of lyophilized and salt-free CAPA powder and normal antigen powder which had been purified according to the previous section, and in this case decreasing pH values were used. The powders were separately dissolved in H ₂ O and the pH was adjusted to 8.5 with 0.1N NH ₄ OH. The pH of the clear solutions was then adjusted to 5.0 with 0.1N HCOOH, which resulted in precipitation. Each precipitate was mixed with 10 ml of polyacrylamide gel slurry which had been equilibrated with an aqueous solution prepared from 0.02 M HCOOH and 0.02 M HCOONH _{4 to} produce a pH of 5.0. Each of these mixtures was applied to a silicone treated 15 ml poly acrylamide column with an inner diameter of 16 mm and a length of 150 mm, which had been equilibrated as above.

A pH gradient was used to elute the precipitates. Using a gradient mixer, the pH was kept at about 5 for a short time to wash the precipitate and the pH was gradually lowered to 2.8. Finally the pH was raised to 9. The buffer system contained 0.02 m HCOH-HCOONH ₄ and 0.02 m NH ₄ HCO ₃ -NH ₃ , and the elution was carried out at room temperature. The flow rate was kept at 27.7 ml / cm ² column cross section per hour.

The leak from the tumor extract was by fluorescence spectrometry (activation at 288 nm, fluorescence at 350 nm emitted) checked. The between the initial pH and a pH of about 3 eluted fraction was used for further Use won. After lyophilization and freezing the product was able to dry in tightly sealed ampoules after removal of air, be stored at -24 ° C.

To characterize the molecular size and confirm the purity, the antigen material was filtered in the form of solutions thereof with polyacrylamide gel (as defined above) which had been equilibrated with a buffer of 0.02 m HCOOH-HCOONH ₄ of 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 / h and the fractions collected were 1.24 ml. The active fractions showed a maximum spectrophotometric absorption at a wavelength of 229 to 233 nm and a fluorescence of 350 nm when at 288 nm were activated.

In a typical experiment, 10.4 mg of CAPA dissolved in 3.0 ml of the above buffer, on a siliconized poly acrylamide gel column (inner diameter 2.54 cm, length 50 cm) for a complete recovery of the substance and the shares vity. After freeze-drying, a white hy was obtained wholesale copy powder in the cold and in the absence of light and oxygen can be stored. With the Analy no carbohydrates and none Nucleic acid found. Analyzes using an amino acid analyzers showed agreement with the molecular ge weight determined by gel filtration. Analyzes of the Amino acids showed the following molar percentages, with the Values have an accuracy of ± 5%. The calculated mole eyepiece weight was 23 200 ± 2500 (standard deviation):

Alanine8,62 Arginine 4.57 Aspartic acid10.39 Cysteine0.95 Glutamic acid 17.04 Glycine 6.87 Histidine 1.00 Isoleucine 4.75 Leucine10.49 Lysine 3,69 Methionine 1.91  Phenylaniline 2.75 Proline3.79 Serine 7.74 Threonine 5.92 Tyrosine 3.21 Valine 6.36

This amino acid content corresponds to a theoretical one Nitrogen content from 16.2 to 17.8%. Determination of the total nitrogen according to Dumas gave a pH of 17.0 ± 0.5%. As demonstrated by treatment with performic acid, be the polypeptide rests on a single peptide chain. Except the polypeptide denatures if not by complex formation is protected with albumin, irreversible at pH-Wer above about 5.

B. Production of Antibodies

Experiments carried out on rabbits showed that paren teral injection of CAPA-albumin complex not to the antibody per formation leads. All hemagglutination reactions were negative. To keep CAPA active in antibody pro to transfer inducing cells, it was necessary to in a solution to pH values below 3 (e.g. 0.02 m HCOOH, pH 2.8) and emulsify this solution in oil to form extremely small drops from one protective oil layer are surrounded. Such an oil emulsion led to the formation of satisfactory amounts of antibody pern that specifically with the CAPA in hemagglutination freak reactions in which blood cells are marked with CAPA are.

816 μg of pure CAPA were dissolved in 1.0 ml of 0.02 m HCOOH of pH 2.8 dissolved. The resulting solution was added dropwise 1.0 ml oil (commercially available Freund's adjuvant) under the same added early emulsification. The emulsification took place after Friend using an injection syringe, taking the mixture in the syringe until a uniform white is formed  Emulsion with about the same consistency as whipped cream was pulled up and down. A drop of this emulsion was tested with ice water to confirm that it was swimming and remained intact. The resulting semi-fluid Emulsion was used for subcutaneous and intramuscular injections used in rabbits.

In the first injection, a so-called complete Excipient used, and in subsequent injections a so-called incomplete auxiliary was used. After the initial bleeding of each of the five rabbits To determine 0 values, a total of 408 micrograms emul CAPA at a pH of 2.8 subcutaneously on each right and left paw area injected. In ten days Intervals were given an additional three injections, the first intramuscularly in each hind leg and the second and third subcutaneously on both front and back The injection sites were selected in order to to use the ducts of the regional lymph glands. Total together, each rabbit received approximately 1.6 mg of CAPA in the form the emulsion. 14 and 24 days after the last injection blood test samples taken from a hemagglutina tion analysis for the presence of specific anti body against CAPA. Two days after that last sample was taken from a maximum amount of blood which was obtained in the form of serum which for its part, sterile filtered and in small portions sterile tube was transferred. These tubes could be stored in the cold, taking into account their specificity stopped.

Claims (2)

1. A process for the preparation of anti-cancer-related antigen (CAPA) monospecific antibodies, wherein in step b) the antigen prepared in step a) is prepared with oil, in particular with Freund's adjuvant, to form an oil emulsion and this then live tie ren injected, characterized in that in step a) an aqueous solution of the antigen with a pH of less than 3 is prepared. 2. The method according to claim 1, characterized in that using the aqueous solution in step a)  a weak acid, preferably formic acid, for acidifying.