DE85693T1 - OBJECTS REGARDING HUMAN INTERFERON ALPHA SUBTYPE PROTEINS AND MATCHING ANTIBODIES. - Google Patents

Claims (1)

-1 - 82 902 WI ". 3 A / S Alfred Benzon Claims 1 · 'HulFN-α proteins, which under SDS PAGE and labeling conditions as defined here, in a complete interferon loading of 5 χ 10 IFU 13 sharp, marked protein bands show the antiviral Have interferon activity at 16600, Ί698Ο, 17380, 17580, 18410, 18840, 19050, 19500, 20420, 20890, 21380, 21880 and 22910 "Daltons", (the "Dalton" molecular weight has a experimental accuracy of -100 "Daltons"), with SDS PAGE marking essentially no other Shows protein region. 2. Single protein that has antiviral interferon activity and is a component of the HulFN-a-interferon ^ 5 proteins according to claim 1 is. 3. Protein with antiviral Interferonaktxvifcät according to claim 2, which appears at 16600-100 "Daltons". 14. Protein according to claim 3, the immunological properties which differ from the miserable remaining 12 specific proteins. 5. Protein, which makes up about 40% of the pure HulFN-a protein, said protein having only negligible activity in the human system and a very high activity in the bovine system and is immunologically different from the HulFN-a proteins, which is a noteworthy Have activity in the human system " 6. Protein with antiviral interferon activity according to claim 2, which appears at 16980 ί 100 "Daltons". 7-protein with antiviral interferon activity according to claim 2 appearing at 17380 t 100 "Daltons". 8. protein having antiviral interferon activity as claimed in claim 2, wherein 17580-100 "Daltons" appears. 9. Protein with antiviral interferon activity according to claim 2, which appears at 18410 ί 100 "Daltons". 10 protein having antiviral interferon activity as claimed in claim 2, wherein 18840-100 "Daltons" appears. 11 protein having antiviral interferon activity as claimed in claim 2, wherein 19050-100 "Daltons" appears. 12 protein having antiviral interferon activity as claimed in claim 2, wherein 19500-100 "Daltons" appears. 13-protein with antiviral interferon activity according to claim 2 appearing at 20420-100 "Daltons". 14. Protein with antiviral interferon activity after an . ■ ■ ■ '' Proverb 2, which appears at 20890-100 "Daltons". 15 protein having antiviral interferon activity as claimed in claim 2, wherein 21380-100 "Daltons" appears. 16. Protein with antiviral interferon activity according to An. Claim 2, which appears at 21880 + 100 "Daltons". 17. Protein with antiviral interferon activity according to claim 2, which appears at 22910 + 100 "Daltons". 18. interferon protein that has a distinct activity in the bovine system and negligible activity in human Systein, wherein said protein at 16600-100 "Daltons" appears. 19. Protein according to claim 1, which has a specific activity 8 Q
did at least 10 to 10 IFU per mg of protein determined by comparative SDS PAGE labeling 20. Proteins according to claim 19 with a specific Aktivi 8 Q
did from 10 to 2 χ 10 IFU per mg of protein, determined by comparative SDS PAGE labeling. 21. Protein (s) according to one of the preceding claims, additionally characterized in that it f 25 has (have) anti-cellular activity and strengthens the natural killer cell system . 22. Protein according to one of claims 6 to 17, additionally characterized in that it is antibodies neutralized against one of the proteins according to one of claims 6 to 17 or against proteins according to Claim 1, excluding the 16600 "Daltons" species. 35 23 · Interferon protein (s) of human leukocytes after a of the preceding claims. 24. Formulation containing a protein or proteins according to any one of the preceding claims, wherein said formulation for use on humans or Animals are suitable for achieving a prophylactic, therapeutic or immunizing effect, 25. Formulation according to claim 24, suitable for parenteral, intranasal or topical application, with or without addition a chemostatically active agent, 26. Formulation according to claim 25 as a cream or ointment for the topical application. 27- formulation according to claim 24 in the form of a stabilized, aqueous solution. 28. Formulation according to claim 27, for the stabilizer is a protein or protein composition that is non-toxic and non-immunizing to humans is. 29. The formulation of claim 28 wherein the stabilizer _s is selected from the group consisting of human serum proteins and fractions thereof, for example menschlichem- albumin or other protein that is not immunogenic in humans. 30. Formulation according to one of claims 24 to 29, in which the concentration of the pure interferon is in a range corresponding to 1 to 40 million IFU per ml. 31- Formulation according to claim 27 for immunizing Animals in which the stabilizer is SDS. 35 32. Formulation according to claim 31, which with PBS in a pH of about 7.2 is buffered. _5- 33. Formulation according to one of claims 31 or 32, the additionally contains an adjuvant. 34. Formulation according to claim 33, in which the adjuvant Freund's adjuvant is. 35. An SDS complex of a protein or proteins according to one of claims 1 to 23- 36. An SDS complex according to claim 35, which is in solid form was isolated. 37 · antibodies raised against immunological determinants of the human Le-form interferon proteins were developed or are essentially only directed against these, according to one of claims 1 to 23- 38. Antibody according to claim 37, which is obtained by immunizing an immunizable animal with one of the 16600, I698O, 17380, 17580, 18410, 18840, 19050, 19500, 20420, 20890, 2138O, 2188O ₅ 229IO "Daltons" HulFN-a components of the proteins according to claim 1 or any combination of these proteins were obtained or by using the same components by means of hybridoma-Teehnik or tear formation technique were obtained. 39- Antibody according to claim 37, which is obtained by immunization of an immunizable animal with one of the I698O, 1738O, 17580, 18410, 18840, 19050, 19500, 20420, 20890, 21380, 21880 22910. 'Daltons' HulFN-a components or proteins Claim 1 or with any composition these have been obtained 40. Antibodies according to claim 37, which are obtained by immunizing an immunizable animal with the 16600 ~ 100 "DaI-tons" HulFN-a component. 41. Antibody according to any one of claims 37 to 40, which is obtained by immunizing an immunizable animal with the HulFN-a component or components from the corresponding band or from the bands from the SDS PAGE-Gel cut, were obtained. 42. Antibody according to claim 41, wherein the bands from the SDS PAGE-Gel after labeling of said gel and brief washing in distilled water to be cut. 43. The method for producing antibodies according to claim 37, wherein an immunizable animal against human Le-form protein or proteins according to any one of claims 2 to 23 is immunized and the antiserum from the Animal is won. 44. The method according to claim 43, wherein the immunization with a protein or proteins according to any one of the claims 2 to 23 is carried out. 45. The method of claim 44, wherein the immunization is carried out with one of the 16600, 16980, 17380, 17580, 18410, 18840, 19050, 19500, 20420, 20890, 21380, 21880, 22910 "Daltons" HulFN-a components of the proteins according to An ^ °° saying 1 or with any combination this. 46. The method of claim 45, wherein the immunization is carried out with one of the 16980, 17380, 17580, 18410, 18840, 21880, 19050, 19500, 20420, 20890, 21380 ,. 22910 "Daltons" HulFN-a components of the proteins according to the ■ Spruch 1 or with any combination of these. 47- The method of claim 45, wherein the immunization performed with the 16600-, 100 "Daltons" HulFN-a component will. P- 48. The method of claim 46, wherein the immunization with the 2188O-100 "Daltons" component is performed. 49. The method according to any one of claims 43 to 48, wherein the Immunization with a formulation according to one of the _n claims 27 and 31 to 34 is carried out. 50. The method for producing antibodies according to claim 37, wherein a hybridoma cell clone is cultivated which Antibodies produced which are directed against immunological determinants of the HulFN-a components according to claim 1 and recovery of the antibodies from the culture medium. 51. Antibodies produced by a method according to any one of claims 43 to 50. 52. Antibodies according to any one of claims 37 to 42 or 51 or fragments or derivatives of these antibodies which retained the essential anti-interferon determinants immobilized on a matrix. 53- The antibody of claim 52, which is covalently attached to the matrix J - P are bound. / -δι 54. Antibody according to claim 53, wherein the matrix is a cross-linked agarose, e.g. Sepharose 4B. 55. Matrix-immobilized antibodies according to one of the claims marriage 52 to 54, which are essentially proteolytic free Are enzyme activity. 56. Matrix-immobilized antibodies according to claim 55, their essential proteolytic enzyme activity thereby removed that they have been treated with enzyme inhibitors or enzyme destructive agents. 57. Matrix-immobilized antibodies according to claim 56, wherein the treatment with enzyme inhibitors or enzymes . destructive agents carried out by matrix-immobilized enzyme inhibitors or enzyme destructive agents will. 58. Matrix-immobilized antibodies according to claim 57, which, prior to their covalent bond to the matrix, through a column will be sent, the matrix-immobilized poly-L-lysine and / or matrix-immobilized soybean trypsin - inhibitor and / or Contains matrix-immobilized kallikrein inactivator. 59- Use of the antibodies according to any one of claims 37 to 42 and 51 to 58 for purifying solutions which contain human le-form interferon, 60. Protein that characterizes the significant interferon Has determinants possessed by the proteins according to any one of claims 1 to 23. 61. Protein that is the significant immunological determinant having the proteins according to any one of claims 1 to 23. 62. Process for making human Le-Form inter- feron proteins according to one of claims 1 or 3 to »; 5, being a solution containing human Le-form interferon proteins contains, is subjected to affinity chromatography i, so that the said human Le- j Form interferon proteins are selectively recovered and said recovered interferon proteins I. be eluted. 63. The method of claim 62, wherein the affinity chromatography consists in that the antibody matrix-immobilized antibodies according to one of claims 52 to 58 are. 64. The method of claim 63, wherein at most 85% of the Antibodies that are applied to the matrix in the immobilized State to be covalently bound. 65. The method of claim 63 or 64, wherein the antibodies prior to being bound to the matrix substantially of any proteolytic enzyme activity by treatment with enzyme inhibitors and / or destructive enzymes Means to be cleaned. 66. The method of claim 65, wherein removing the proteolytic activity with a matrix-immobilized Enzyme inhibitor and / or enzyme-destroying agents is carried out. 67. The method according to any one of claims 62 to 66, wherein the solution applied to the antibody affinity matrix is a crude, unfocused interferon preparation. 68. The method according to any one of claims 62 to 66, wherein the Solution ^ which is added to the antibody affinity matrix "* is a concentrated or partially purified interferon preparation.. '. * 0085693 -ιοί 69. The method according to any one of claims 62 to 66, wherein the interferon-containing solution is the interferon-containing protein fraction> that by protein precipitation treatment of a raw, unfocused Interferon preparation was obtained, 70. The method of claim 69, wherein the protein precipitation treatment includes adding KSCN and adjusting the solution to pH 4.5. 71. The method according to any one of claims 62 to 70, wherein the solution applied to the antibody affinity matrix is an interferon solution which essentially only contains proteins in the 10,000 to 20,000 Dalton range. 72. The method of claim 71, wherein the solution is the 10,000 to 20,000 Dalton eluate from a gel filtration which is carried out with a buffer solution containing about 25% by volume of a glycol, for example ethylene glycol and has an ionic strength corresponding to 1 M NaCl, pH about 7.2. 73. The method according to any one of claims 63 to 72, wherein the HulFN-a interferon of the solution that the antibody affini « ity matrix is selected from a group containing: human leukocyte interferons, ■ human lymphoblast interferons and proteins according to any one of claims 60 or 61, including those proteins obtained by culturing a microorganism which contains a DNA which codes for the production of such a protein. 74. The method of claim 73, wherein the interferon-containing Solution that contains the protein fraction containing interferon, which by protein precipitation treatment a raw, unfocused interferon preparation is obtained. ■ 75. The method of claim 74, wherein the protein precipitation treatment includes adding KSCN and adjusting the solution to pH 4.5. 76. The method according to any one of claims 73 to 75, wherein the the solution applied to the antibody affinity matrix Interferon solution is essentially just proteins contains in the 10,000 to 20,000 Dalton range. 77. The method of claim 76, wherein the solution comprises 10,000 is up to 20,000 Dalton eluate of a gel filtration with a buffer solution is carried out which contains about 25% by volume of a glycol, for example ethylene glycol and an ionic strength corresponding to 1 M MaCl, pH about 7.2, has. 78. The method according to any one of claims 74 to 77, wherein the antibody affinity matrix is loaded directly with the eluate in the gel filtration buffer. _: 79. HulFN-a interferon produced by the use of matrix-immobilized antibodies according to any one of claims 52 to 58 or by a method according to any one of claims 62 to 78. 80. HulFN-a interferon according to claim 79, which has a specific Has activity of at least 30 χ 10 IFU per mg protein, the determination of the protein being based on the Lowry method using pure human albumin serum as the standard. ■ 81. HulFN-a interferon according to claim 80 having a specific active
mg protein. r Q see activity in the range of 30 χ 10 to 10 IFU per 82. HulFN-a interferon according to claim 81, with a specific activity of 30 χ 10 to 7Ox 10 IFU per mg of protein . 83- Method of making interferon proteins or Proteins that are the significant determinants of interferon biological activity of these proteins by cultivating a microorganism that has a Carries DNA necessary for the production of HulFN-interferon proteins according to any one of claims 1 to 23 or 60 and said proteins from the culture medium be won. 84, The method of claim 83, wherein the DNA coding for the production of the interferon proteins on a Plasmid lies. 85. The method according to claim 83 or 84, wherein the DNA which codes for the production of the interferon proteins is produced by treatment with reverse transcriptase of a messenger RNA isolated from interferon-producing cells, the method being known per se and wherein the method further contains the stage of antibody affinity chromatography and / or immunoprecipitation of a lysate of the cells and the antibodies which are used in antibody affinity chromatography or in immunoprecipitation, antibodies according to any one of claims 37 to 42 or 51 are. 86. The method of claim 83 to 85, wherein the interferon producing clones of the microorganism by means of radiolabeling monospecific antibodies according to one of the Claims 37 to 42 or 51 are selected.