IPC분류정보
국가/구분 |
United States(US) Patent
등록
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국제특허분류(IPC7판) |
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출원번호 |
UP-0714228
(2003-11-14)
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등록번호 |
US-7754450
(2010-08-02)
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발명자
/ 주소 |
- Grasso, Luigi
- Liang, Shaohong
- Nicolaides, Nicholas C.
- Sass, Philip M.
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 |
피인용 횟수 :
3 인용 특허 :
172 |
초록
▼
The invention provides methods for generating high titers of high-affinity antibodies from hybridoma cells produced by fusing myeloma cells with in vitro immunized donor cells. The hybridoma cells or mammalian expression cells with cloned antibody genes from the hybridomas producing the high-affinit
The invention provides methods for generating high titers of high-affinity antibodies from hybridoma cells produced by fusing myeloma cells with in vitro immunized donor cells. The hybridoma cells or mammalian expression cells with cloned antibody genes from the hybridomas producing the high-affinity antibodies may be mismatch repair defective due to defects of endogenous mismatch repair subunits of through expression of a dominant negative allele of a mismatch repair gene which allows the hybridoma cell to be hypermutable, may be rendered hypermutable by chemical means, or may be naturally mismatch repair deficient. High-affinity antibodies and high titer producer cells producing antibodies may be prepared by the methods of the invention.
대표청구항
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What is claimed is: 1. A method for producing hybridoma cells producing antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing ce
What is claimed is: 1. A method for producing hybridoma cells producing antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells, (e) screening antibodies produced from said hypermutated hybridoma cells for binding to antigen; and (f) selecting hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells; thereby producing hybridoma cells producing antibodies having higher affinity for said antigen than antibodies produced by said parental hybridoma cells. 2. The method of claim 1 wherein said chemical inhibitor of mismatch repair is an anthracene, ATPase inhibitor, a nuclease inhibitor, an RNA interference molecule, a polymerase inhibitor, or an antisense oligonucleotide that specifically hybridizes to a nucleotide encoding a mismatch repair protein. 3. A method for producing hybridoma cells producing antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula: wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups; (d) screening antibodies produced from said hypermutated hybridoma cells for binding to antigen; and (e) selecting hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells; thereby producing hybridoma cells producing antibodies having higher affinity for said antigen than antibodies produced by said parental hybridoma cells. 4. The method of claim 3 wherein R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. 5. The method of claim 1 further comprising screening for hypermutated hybridoma cells that also produce antibodies in higher titers than said parental hybridoma cells. 6. The method of claim 3 further comprising the step of removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells. 7. The method of claim 1 wherein said antibodies having higher affinity for said antigen than antibodies produced by said parental hybridoma cells have an affinity for said antigen of at least about 1×107 M−1 to about 1×1014 M−1. 8. The method of claim 5 wherein said hypermutated hybridoma cells that produce antibodies in higher titers than said parental hybridoma cells have a titer that is at least about 1.5-8 fold greater than the titer produced by said parental hybridoma cells. 9. A method for producing hybridoma cells that produce antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce higher titers of antigen-specific antibodies than said parental hybridoma cells; thereby producing hybridoma cells that produce higher titers of antibodies than said parental hybridoma cells. 10. The method of claim 9 wherein said chemical inhibitor of mismatch repair is an anthracene, ATPase inhibitor, a nuclease inhibitor, an RNA interference molecule, a polymerase inhibitor, or an antisense oligonucleotide that specifically hybridizes to a nucleotide encoding a mismatch repair protein. 11. A method for producing hybridoma cells that produce antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula: wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups; and (d) selecting hypermutated hybridoma cells that produce higher titers of antigen-specific antibodies than said parental hybridoma cells; thereby producing hybridoma cells that produce higher titers of antibodies than said parental hybridoma cells. 12. The method of claim 11 wherein R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. 13. The method of claim 11 further comprising the step of removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells. 14. The method of claim 9 wherein said higher titer of said antibodies is a titer of at least about 1.5-8 fold greater than the titer of said parental hybridoma cells. 15. A method for producing mammalian expression cells that produce antibodies comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; (d) cloning immunoglobulin genes from hybridoma cells that produce antibodies to said antigen into a mammalian expression cell; (e) incubating said mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated mammalian expression cells; (t) removing said chemical inhibitor of mismatch repair from said hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells; and (g) performing a screen for hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said hybridoma cells that produce antibodies to said antigen; thereby producing mammalian expression cells that produce antibodies having higher affinity for said antigen than said hybridoma cells that produce antibodies to said antigen. 16. The method of claim 15 wherein said chemical inhibitor of mismatch repair is an anthracene, ATPase inhibitor, a nuclease inhibitor, an RNA interference molecule, a polymerase inhibitor, or an antisense oligonucleotide that specifically hybridizes to a nucleotide encoding a mismatch repair protein. 17. A method for producing mammalian expression cells that produce antibodies comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; (d) cloning immunoglobulin genes from hybridoma cells that produce antibodies to said antigen into a mammalian expression cell; (e) incubating said mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated mammalian expression cells, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula: wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups; and (f) performing a screen for hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said hybridoma cells that produce antibodies to said antigen; thereby producing mammalian expression cells that produce antibodies having higher affinity for said antigen than said hybridoma cells that produce antibodies to said antigen. 18. The method of claim 17 wherein R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. 19. The method of claim 15 further comprising performing a screen for hypermutated mammalian expression cells that also produce antibodies in higher titers than said hybridoma cells that produce antibodies to said antigen. 20. The method of claim 15 wherein said antibodies having higher affinity for said antigen than said hybridoma cells that produce antibodies to said antigen have an affinity for said antigen of at least about 1×107 M−1 to about 1×1014 M−1. 21. The method of claim 19 wherein said higher titers of said antibodies is at least about 1.5-8 fold greater that the titer produced by said hybridoma cells that produce antibodies to said antigen. 22. The method of claim 17 further comprising removing said chemical inhibitor of mismatch repair from said hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells. 23. A method for producing mammalian expression cells that produce antibodies to a selected antigen from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells; (d) removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells; (e) performing a screen for binding of antigen for antibodies produced from said hypermutated hybridoma cells; (f) selecting hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells; (g) cloning immunoglobulin genes from said hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells into a mammalian expression cell, thereby forming parental mammalian expression cells; thereby producing mammalian expression cells that produce antibodies having higher affinity for said antigen than said parental hybridoma cells from in vitro immunized immunoglobulin-producing cells. 24. The method of claim 23 wherein said chemical inhibitor of mismatch repair is an anthracene, ATPase inhibitor, a nuclease inhibitor, an RNA interference molecule, a polymerase inhibitor, or an antisense oligonucleotide that specifically hybridizes to a nucleotide encoding a mismatch repair protein. 25. A method for producing mammalian expression cells that produce antibodies to a selected antigen from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula: wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups; (d) performing a screen for binding of antigen for antibodies produced from said hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells; and (f) cloning immunoglobulin genes from said hypermutated hybridoma cells that produce antibodies with higher affinity for said antigen than antibodies produced by said parental hybridoma cells into a mammalian expression cell, thereby forming parental mammalian expression cells; thereby producing mammalian expression cells that produce antibodies having higher affinity for said antigen than said parental hybridoma cells from in vitro immunized immunoglobulin-producing cells. 26. The method of claim 25 wherein R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. 27. The method of claim 23 wherein said antibodies having higher affinity for said antigen than said parental hybridoma cells have an affinity for said antigen of at least about 1×107M−1 to about 1×1014 M−4. 28. The method of claim 23 further comprising the steps of: incubating said parental mammalian expression cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated mammalian expression cells; and screening for hypermutated mammalian expression cells that produce a higher titer of antibodies than said parental mammalian expression cells. 29. The method of claim 25 further comprising removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells. 30. The method of claim 28 further comprising removing said chemical inhibitor of mismatch repair from said hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells. 31. The method of claim 28 wherein said higher titer of antibodies is at least about 1.5-8 fold greater than the titer produced by said parental mammalian expression cells. 32. The method of claim 1, 9, 15 or 23 wherein said chemical inhibitor of mismatch repair is an antisense molecule comprising at least 15 consecutive nucleotides of a sequence encoding a protein selected from the group consisting of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; and SEQ ID NO:50. 33. The method of claim 1, 9, 15 or 23 wherein said chemical inhibitor of mismatch repair is an antisense molecule comprising at least 15 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; and SEQ ID NO:49. 34. The method of claim 25 wherein said antibodies having higher affinity for said antigen than said parental hybridoma cells have an affinity for said antigen of at least about 1×107M−1 to about 1×1014 M−1. 35. The method of claim 25 further comprising the steps of: incubating said parental mammalian expression cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated mammalian expression cells; and screening for hypermutated mammalian expression cells that produce a higher titer of antibodies than said parental mammalian expression cells. 36. The method of claim 35 further comprising removing said chemical inhibitor of mismatch repair from said hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells. 37. The method of claim 35 wherein said higher titer of antibodies is at least about 1.5-8 fold greater than the titer produced by said parental mammalian expression cells. 38. The method of claim 17 further comprising performing a screen for hypermutated mammalian expression cells that also produce antibodies in higher titers than said hybridoma cells that produce antibodies to said antigen. 39. The method of claim 17 wherein said antibodies having higher affinity for said antigen than said hybridoma cells that produce antibodies to said antigen have an affinity for said antigen of at least about 1×107M−1 to about 1×1014 M−1. 40. The method of claim 38 wherein said higher titers of said antibodies is at least about 1.5-8 fold greater that the titer produced by said hybridoma cells that produce antibodies to said antigen. 41. The method of claim 11 wherein said higher titer of said antibodies is a titer of at least about 1.5-8 fold greater than the titer of said parental hybridoma cells. 42. The method of claim 3 further comprising screening for hypermutated hybridoma cells that also produce antibodies in higher titers than said parental hybridoma cells. 43. The method of claim 42 further comprising removing said chemical inhibitor of mismatch repair from said hypermutated hybridoma cells, thereby stabilizing the genome of said hypermutated hybridoma cells. 44. The method of claim 3 wherein said antibodies having higher affinity for said antigen than antibodies produced by said parental hybridoma cells have an affinity for said antigen of at least about 1×107 M−1 to about 1×1014 M−1. 45. The method of claim 42 wherein said hypermutated hybridoma cells that produce antibodies in higher titers than said parental hybridoma cells have a titer that is at least about 1.5-8 fold greater than the titer produced by said parental hybridoma cells.
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