Methods for generating genetically altered antibody-producing cell lines with improved antibody characteristics 机翻标题: 暂无翻译,请尝试点击翻译按钮。

公开号/公开日
CA2428214 A1 2002-05-16 [CA2428214]CA2428214 C 2010-02-02 [CA2428214] / 2002-05-162010-02-02
申请号/申请日
2000CA-2428214 / 2000-11-07
发明人
NICOLAIDES NICHOLAS C;GRASSO LUIGI;SASS PHILIP M;
申请人
MORPHOTEK;
主分类号
IPC分类号
A61K-039/395A61K-048/00C07H-021/04C07K-016/00C07K-016/42C12N-005/10C12N-015/00C12N-015/09C12N-015/10C12P-021/06C12P-021/08C12Q-001/68
摘要
(CA2428214) Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms.  By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on th e natural rate of mutation.  These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interes t to produce altered antibodies with enhanced biochemical activity.  Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production.
机翻摘要
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地址
代理人
代理机构
;
优先权号
2000WO-US30588 2000-11-07
主权利要求
(CA2428214)  CLAIMS 1. A method for making a hypermutable, antibody producing cell, comprising introducing into a cell capable of producing antibodies a polynucleotide comprising a dominant negative allele of a mismatch repair gene, wherein said mismatch repair gene is PMS2. 2. The method of claim 1 wherein said mismatch repair gene is human PMS2. 3. The method of claim 2 wherein said allele comprises a truncation mutation. 4. The method of claim 2 wherein said allele comprises a truncation mutation at codon 134. 5. The method of claim 1 wherein said capability is due to the cointroduction of an immunoglobulin gene into said cell. 6. A method for generating a mutation in an antibody-producing cell, the mutation causing overexpression, enhanced secretion, or enhanced affinity for antigen of an antibody produced by the cell, comprising: growing said antibody-producing cell comprising a dominant negative allele of a mismatch repair gene, wherein said mismatch repair gene is PMS2;  and testing the cell for overexpression, enhanced secretion, or enhanced affinity for an antigen of an antibody produced by the cell. 7. A method of reversibly altering the hypermutability of an antibody producing cell comprising introducing an inducible vector into a cell, wherein said inducible vector comprises a dominant negative allele of a PMS2 mismatch repair gene operably linked to an inducible promoter, and inducing said cell to express said dominant negative allele. 8. The method of claim 7 wherein said mismatch repair gene is human PMS2. 9. The method of claim 7 further comprising ceasing induction of said cell, thereby restoring genetic stability of said cell. 10. A method of producing a genetically altered antibody comprising: transfecting a polynucleotide encoding an immunoglobulin protein into a cell, wherein said cell comprises a dominant negative allele of a mismatch repair gene, wherein said mismatch repair gene is PMS2;  growing said cell, thereby producing a hypermutated polynucleotide encoding a hypermutated immunoglobulin protein;  screening for a property of said hypermutated immunoglobulin protein;  isolating said hypermutated polynucleotide;  and transfecting said hypermutated polynucleotide into a genetically stable cell, and growing said cell under suitable conditions to produce said genetically altered antibody. 11. The method of claim 10 wherein said mismatch repair gene is human PMS2. 12. The method of claim 10 wherein said cell expresses a protein consisting of the first 133 amino acids of hPMS2. 13. The method of claim 1 further comprising the step of restoring genetic stability of said hypermutable cell. 14. A method of producing a genetically stable, antibody-producing cell comprising: transfecting a polynucleotide encoding an immunoglobulin protein into a cell, wherein said cell comprises a dominant negative allele of a mismatch repair gene wherein said mismatch repair gene is PMS2;  growing said cell, thereby producing a hypermutated polynucleotide encoding a hypermutated immunoglobulin protein;  screening for a property of said hypermutated immunoglobulin protein;  isolating said hypermutated polynucleotide;  and transfecting said hypermutated polynucleotide into a genetically stable cell, thereby producing a hypermutated antibody-producing, genetically stable cell. 15. The method of claim 10 further comprising recovering said genetically altered antibody. 16. A homogeneous culture of isolated, hypermutable, mammalian cells wherein said cells produce antibodies and comprise a dominant negative allele of a mismatch repair gene, wherein said mismatch repair gene is PMS2. 17. The culture of isolated, hypermutable, mammalian cells of claim 16 wherein the mismatch repair gene is human PMS2. 18. The culture of isolated, hypermutable, mammalian cells of claim 16 wherein the cells express a protein comprising the first 133 amino acids of PMS2. 19. The method of claim 6 wherein said mismatch repair gene is human PMS2. 20. The method of claim 6 wherein said dominant negative allele comprises a truncation mutation. 21. The method of claim 6 wherein said dominant negative allele comprises a truncation mutation at codon 134. 22. The method of claim 6 wherein said cell expresses a protein consisting of the first 133 amino acids of hPMS2. 23. The method of claim 7 wherein said dominant negative allele comprises a truncation mutation. 24. The method of claim 7 wherein said dominant negative allele comprises a truncation mutation at codon 134. 25. The method of claim 7 wherein said cell expresses a protein consisting of the first 133 amino acids of hPMS2. 26. The method of claim 7 wherein said dominant negative allele comprises a truncation mutation. 27. The method of claim 7 wherein said dominant negative allele comprises a truncation mutation at codon 134. 28. The method of claim 14 wherein said mismatch repair gene is human PMS2. 29. The method of claim 14 wherein said dominant negative allele comprises a truncation mutation. 30. The method of claim 14 wherein said dominant negative allele comprises a truncation mutation at codon 134. 31. The method of claim 14 wherein said cell expresses a protein consisting of the first 133 amino acids of hPMS2. 32. A method for generating a library of cells producing mutated antibodies, comprising: expressing in antibody-producing cells a dominant negative allele of a PMS2 mismatch repair gene, wherein said expression inhibits mismatch repair of said antibody-producing cells, and selecting cells that produce a mutated antibody, thereby producing a library of cells producing mutated antibodies. 33. The method of claim 32 further comprising restoring mismatch repair activity to said cells that produce a mutated antibody. 34. The method of claim 32 further comprising a step of expressing a polynucleotide encoding said mutated antibody in a genetically stable cell. 35. A method for generating a library of mutated antibody-producing cells comprising expressing in antibody-producing cells a dominant negative allele of a PMS2 mismatch repair gene, wherein said expression inhibits mismatch repair of said antibody-producing cells, thereby producing a library of mutant antibodyproducing cells. 36. The method of claim 35 further comprising restoring mismatch repair activity to said mutant antibody-producing cells. 37. A method for generating an antibody-producing cell line comprising a mutation in a gene of interest comprising: introducing a dominant negative allele of a PMS2 mismatch repair gene in a population of antibody-producing cells in culture, wherein said antibodyproducing cells comprise said gene of interest, thereby inhibiting mismatch repair in said antibody-producing cells, separating said population into individual members of the population, identifying members of the population comprising a mutation in the gene of interest, restoring mismatch repair activity in said members of said population comprising a mutation in the gene of interest, and expanding said members comprising a mutation in the gene of interest, thereby generating an antibody-producing cell line comprising a mutation in a gene of interest. 38. The method of claim 37 wherein said gene of interest encodes an antibody. 39. A method for generating an antibody-producing cell line comprising a mutation in a gene of interest comprising: introducing a dominant negative allele of a PMS2 mismatch repair gene in a population of antibody-producing cells in culture, wherein said antibodyproducing cells comprise said gene of interest, thereby inhibiting mismatch repair in said antibody-producing cells, separating said population into individual members of the population, identifying members of the population comprising a mutation in the gene of interest, expressing a polynucleotide comprising a mutation in the gene of interest in a genetically stable cell, and expanding said genetically stable cell, thereby generating an antibody-producing cell line comprising a mutation in a gene of interest. 40. The method of claim 39 wherein said gene of interest encodes an antibody. 41. A method for generating a library of mutated antibodies, comprising: expressing in antibody-producing cells a dominant negative allele of a PMS2 mismatch repair gene, selecting cells that produce a mutated antibody, and isolating mutated antibodies, thereby producing a library of mutated antibodies. 42. The method of claim 41 further comprising restoring mismatch repair activity to said cells that produce a mutated antibody.
法律状态
(CA2428214) LEGAL DETAILS FOR CA2428214  Actual or expected expiration date=2020-11-07    Legal state=ALIVE    Status=GRANTED     Event publication date=2000-11-07  Event code=CA/APP  Event indicator=Pos  Event type=Examination events  Application details  Application country=CA CA2428214  Application date=2000-11-07  Standardized application number=2000CA-2428214     Event publication date=2002-05-16  Event code=CA/A1  Event type=Examination events  Application laid open  Publication country=CA  Publication number=CA2428214  Publication stage Code=A1  Publication date=2002-05-16  Standardized publication number=CA2428214     Event publication date=2005-11-07  Event code=CA/EEER  Event indicator=Pos  Event type=Examination events  Examination request    Event publication date=2010-02-02  Event code=CA/C  Event indicator=Pos  Event type=Event indicating In Force  Patent (second level)  Publication country=CA  Publication number=CA2428214  Publication stage Code=C  Publication date=2010-02-02  Standardized publication number=CA2428214
专利类型码
A1C
国别省市代码
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