Cancer associated polypeptide antigen, process for its preparation, process for preparing antibodies, process of cancer diagnosis and composition useful as an immunizing agent

Claim 2

A process for the isolation of a cancer associated polypeptide antigen (CAPA) comprising the steps: a. homogenizing CAPA-containing material in a liquid at a temperature not above about 0°C. b. separating solids from the mixture of liquid homogenate and solids results from step a) c. extracting said solids with an alkaline aqueous solution; c'. heat-stabilizing the product by rapidly heating to a temperature range between about 95° and about 100° C. to destroy enzymes destructive to the CAPA; d. filtering the extract resulting from step c') on a filtration gel of the molecular sieve type in bead form permitting fractionation of the compounds having a molecular weight range including 10.sup.. 10 6 to 20.sup.. 10 6 , especially about 15.sup.. 10 6 , eluting the gel and recovering the 10.sup.. 10 6 to 15.sup.. 10 6 to 15.sup.. 10 6 fraction, especially the fraction of about 15.sup.. 10 6 d'. dissolving the sedimented active fractions from the gel filtration in a buffer of pH about 7; e. subjecting the fraction resulting from step d') to chromatography on a weak ion exchanger equilibrated with a buffer of pH about 7 to adsorb thereon impurities and recovering the first fraction leaving said exchanger; f. subjecting said first fraction to isoelectric precipitation, f'. subjecting the fractions from the isoelectric precipitation to active-substance precipitation from aqueous solution at pH about 5, and dissolving the powder in water at pH about 8.5 and acidifying the clear solution with formic acid to produce a precipitate, and thereafter g. mixing the resulting precipitate with a molecular sieve type gel and layering the mixture on top of a column of an identical molecular sieve type gel of the same isoelectric pH and subjecting said gel to pH gradient elution under gradually decreasing pH; and h. while decreasing the pH, recovering the eluate fraction down to pH about 3 containing CAPA therein having a spectrophotometric absorption peak wave length at 229-233 nm and a molecular weight within the range of about 20,000 - 27,000.

Claim 3

A process according to claim 1, wherein the fraction resulting from the pH-gradient elution is subjected to gel filtration on a gel column, the eluted fractions of antigen having a spectrophotometric absorption peak wave length at 229-233 nm and a molecular weight within the range of about 20,000 - 27,000 being recovered.

Claim 4

A process according to claim 1, wherein the homogenate resulting from step a) is subjected to organic solvent treatment in the cold before step b).

Claim 5

A process according to claim 3, wherein the solvent used is ethyl ether.

Claim 6

A process according to claim 1, wherein the homogenization is carried out in water at a temperature not above 0°C.

Claim 7

A process according to claim 1, wherein the solids resulting from step b) are lyophilized and then ground in a steel ball mill in the cold.

Claim 8

A process according to claim 6 wherein the grinding takes place at low temperature.

Claim 9

A process according to claim 6, wherein the grinding takes place at a temperature below the freezing point of the material under treatment.

Claim 10

A process according to claim 6, wherein the grinding takes place at a temperature of about --70° C.

Claim 11

A process according to claim 1, wherein under step d) the filtering is carried out on a gel selected from the group consisting of polyacrylamide gels, cross-linked dextran gels, agar and agarose gels, the gel being equilibrated with phosphate buffer at about pH 7.

Claim 12

A process according to claim 1, wherein under step e) the chromatography is carried out on a column of a weak ion exchanger at neutral pH.

Claim 13

A process according to claim 11, wherein the chromatography is carried out on a weak ion exchanger polyacrylamide gel, said gel being in the form of a column at a neutral pH.

Claim 14

A process according to claim 1, wherein under step g) there is used a gel selected from the group consisting of polyacrylamide gels, cross-linked dextran gels, agar and agarose gels.

Claim 15

A process according to claim 2, wherein said gel filtration is carried out on a gel selected from the group consisting of polyacrylamide gel and cross-linked dextran gel.

Claim 16

A process according to claim 1, wherein said aqueous extract is rapidly heated to a temperature within the range of about 98°-100°C and maintained at said temperature for about 5-10 minutes.

Claim 17

A process for the preparation of CAPA comprising: homogenizing antigen-containing material while still in the frozen state, in water at a temperature not above about 0°C; subjecting the resulting homogenate to treatment with a solvent selected from the group consisting of ethyl ether and acetone; separating solids from the resulting mixture of liquid homogenate and solids; lyophilizing the resulting solids and grinding same in a steel ball mill at a temperature below the freezing point of the material under treatment; extracting the resulting solids with an alkaline aqueous solution and, after centrifugation, subjecting the supernatant liquid to rapid heating to a temperature within the range of about 95 to about 100°C for a period of about 5 to about 10 minutes to destroy enzymes; treating the heated supernatant with an aqueous sodium chloride solution to precipitate contaminants and recovering the supernatant liquid; filtering and resulting liquid on a cold column loaded with a molecular sieve type gel in bead form having a fractionation range of about 10,000 to at least 20.sup.. 10 6 and equilibrated with phosphate buffer at about pH 7.0 and recovering the active fraction of the eluate; subjecting said mid-fraction to chromatography on a column loaded with a weak ion exchanger molecular sieve type gel equilibrated with phosphate buffer of about pH 7.0 and recovering the first fraction of the liquid leaving the column; subjecting said first fraction to isoelectric precipitation at about pH 5 and mixing the resulting precipitate with a molecular sieve type gel having a molecular weight exclusion limit of at least about 2000 equilibrated with HCOOH-HCOONH 4 at about pH 5, applying the resulting mixture on top of a column loaded with an identical gel equilibrated in the same way, pH-gradient eluting the column and, while decreasing the pH, recovering the fraction eluted down to about pH 3 containing the CAPA having a spectrophotometric absorption peak wave length at 229-233 nm and a molecular weight of 23,200 ± 2,500 (S.D.).

Claim 18

A process for the preparation of CAPA comprising: homogenizing antigen-containing material while still in the frozen state, in water at a temperature not above about 0°C; subjecting the resulting homogenate to treatment with a solvent selected from the group consisting of ethyl ether and acetone; separating solids from the resulting mixture of liquid homogenate and solids; lyophilizing the resulting solids and grinding same in a steel ball mill at a temperature below the freezing point of the material under treatment; extracting the resulting solids with an alkaline aqueous solution and, after centrifugation, subjecting the supernatant liquid to rapid heating to a temperature within the range of about 95 to about 100°C for a period of about 5 to about 10 minutes to destroy enzymes; treating the heated supernatant with an aqueous sodium chloride solution to precipitate contaminants and recovering the supernatant liquid; filtering and resulting liquid on a cold column loaded with a molecular sieve type gel in bead form having a fractionation range of about 10,000 to at least 20.sup.. 10 6 and equilibrated with phosphate buffer at about pH 7.0 and recovering the active fraction of the eluate; subjecting said mid-fraction to chromatography on a column loaded with a weak ion exchanger molecular sieve type gel equilibrated with phosphate buffer of about pH 7.0 and recovering the first fraction of the liquid leaving the column; subjecting said first fraction to isoelectric precipitation at about pH 5 and mixing the resulting precipitate with a molecular sieve type gel having a molecular weight exclusion limit of at least about 2000 equilibrated with HCOOH-HCOONH 4 at about pH 5, applying the resulting mixture on top of a column loaded with an identical gel equilibrated in the same way, pH-gradient eluting the column and, while decreasing the pH, recovering the fraction eluted down to about pH 3 containing the CAPA having a spectrophotometric absorption peak wave length at 229-233 nm and a molecular weight of 23,200 ± 2,500 (S.D.).

Claim 19

A process according to claim 1, wherein said antigen-containing material is tissue from human placenta.

Claim 20

A process according to claim 1, wherein said antigen-containing material is human tumor tissue.

Claim 21

A process according to claim 1, wherein said antigen-containing material is a culture of cancerous cells grown in vitro.