USRE48651EActiveUtility

Production cell line enhancers

74
Assignee: REGENERON PHARMAPriority: May 29, 2012Filed: Apr 17, 2019Granted: Jul 20, 2021
Est. expiryMay 29, 2032(~5.9 yrs left)· nominal 20-yr term from priority
Inventors:Gang ChenDarya BurakovDipali Deshpande
C07K 16/22C12N 5/16C12N 2510/00C12N 15/85C12N 9/2402C07K 2317/14C07K 14/4702C12Y 302/01C07K 2317/51C07K 16/00C07K 14/47C12N 9/2488C07K 2317/515C12N 15/79C07K 14/4705C12N 15/63C12N 2015/8518C12N 15/09
74
PatentIndex Score
0
Cited by
70
References
38
Claims

Abstract

The present invention relates to discovery of the ectopic expression of EDEM2 in a production cell to improve the yield of a useful multi-subunit protein. Thus, the present invention provides for production cell lines, such as the canonical mammalian biopharmaceutical production cell—the CHO cell, containing recombinant polynucleotides encoding EDEM2. Also disclosed is a production cell containing both an EDEM2-encoding polynucleotide as well an XBP1-encoding polynucleotide. Improved titers of antibodies produced by these cell lines are disclosed, as well as the improved cell densities attained by these cells in culture.

Claims

exact text as granted — not AI-modified
What is claimed: 
     
       1. An isolated polynucleotide comprising a nucleotide sequence, which encodes an endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2), a promoter, and a multi-subunit protein, wherein the EDEM2 comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 1, and wherein the multi-subunit protein comprises at least one antibody heavy chain antibody and or at least one antibody light chain antibody. 
     
     
       2. The isolated polynucleotide of  claim 1 , wherein the promoter is selected from the group consisting of ubiquitin C, CMV-IE and SV40. 
     
     
       3. The isolated polynucleotide of  claim 2 , wherein the EDEM2 comprises the amino acid sequence of SEQ ID NO: 8. 
     
     
       4. The isolated polynucleotide of  claim 2 , wherein the EDEM2 consists essentially of the amino acid sequence of SEQ ID NO: 8. 
     
     
       5. The isolated polynucleotide of  claim 2 , wherein the EDEM2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
       6. The isolated polynucleotide of  claim 2 , wherein the EDEM2 consists essentially of the amino acid sequence of SEQ ID NO: 1. 
     
     
       7. The isolated polynucleotide of  claim 1 , wherein the nucleotide sequence further multi-subunit protein comprises at least one antibody heavy chain or and at least one antibody light chain. 
     
     
       8. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes the heavy chain comprising amino acid sequences SEQ ID NO: 43 and SEQ ID NO: 44 and the light chain comprising amino acid sequences SEQ ID NO: 45 and SEQ ID NO: 46. 
     
     
       9. The isolated polynucleotide of  claim 7 , where the nucleotide sequence encodes an anti-GDF8 antibody heavy chain, operably linked to a mammalian ubiquitin C promoter or a human CMV-IE promoter. 
     
     
       10. The isolated polynucleotide of  claim 9 , wherein the anti-GDF8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 20. 
     
     
       11. The isolated polynucleotide of  claim 9 , wherein the anti-GDF8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 19. 
     
     
       12. The isolated polynucleotide of  claim 9  comprising the nucleotide sequence of SEQ ID NO: 23. 
     
     
       13. The isolated polynucleotide of  claim 9  comprising the nucleotide sequence of SEQ ID NO: 24. 
     
     
       14. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes an anti-GDF8 antibody light chain, operably linked to a mammalian ubiquitin C promoter or a human CMV-IE promoter. 
     
     
       15. The isolated polynucleotide of  claim 14 , wherein the anti-GDF8 antibody light chain comprises the amino acid sequence of SEQ ID NO: 22. 
     
     
       16. The isolated polynucleotide of  claim 14 , wherein the anti-GDF8 antibody light chain comprises the amino acid sequence of SEQ ID NO: 21. 
     
     
       17. The isolated polynucleotide of  claim 14  comprising the nucleotide sequence of SEQ ID NO: 25. 
     
     
       18. The isolated polynucleotide  claim 14  comprising the nucleotide sequence of SEQ ID NO: 26. 
     
     
       19. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes an anti-ANG2 antibody heavy chain, operably linked to a mammalian ubiquitin C promoter or a human CMV-IE promoter. 
     
     
       20. The isolated polynucleotide of  claim 19 , wherein the anti-ANG2 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 28. 
     
     
       21. The isolated polynucleotide of  claim 19 , wherein the anti-ANG2 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
       22. The isolated polynucleotide of  claim 19  comprising the nucleotide sequence of SEQ ID NO: 31. 
     
     
       23. The isolated polynucleotide of  claim 19  comprising the nucleotide sequence of SEQ ID NO: 32. 
     
     
       24. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes an anti-ANG2 antibody light chain, operably linked to a promoter. 
     
     
       25. The isolated polynucleotide of  claim 24 , wherein the anti-ANG2 antibody light chain comprises the amino acid sequence of SEQ ID NO: 30. 
     
     
       26. The isolated polynucleotide of  claim 24 , wherein the anti-ANG2 antibody light chain comprises the amino acid sequence of SEQ ID NO: 29. 
     
     
       27. The isolated polynucleotide of  claim 24  comprising the nucleotide sequence of SEQ ID NO: 33. 
     
     
       28. The isolated polynucleotide of  claim 24  comprising the nucleotide sequence of SEQ ID NO: 34. 
     
     
       29. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes an anti-AngPtl4 antibody heavy chain, operably linked to a mammalian ubiquitin C promoter or a human CMV-IE promoter. 
     
     
       30. The isolated polynucleotide of  claim 29 , wherein the anti-AngPtl4 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 36. 
     
     
       31. The isolated polynucleotide of  claim 29 , wherein the anti-AngPtl4 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 35. 
     
     
       32. The isolated polynucleotide of  claim 29  comprising the nucleotide sequence of SEQ ID NO: 39. 
     
     
       33. The isolated polynucleotide of  claim 29  comprising the nucleotide sequence of SEQ ID NO: 40. 
     
     
       34. The isolated polynucleotide of  claim 7 , wherein the nucleotide sequence encodes an anti-AngPtl4 antibody light chain, operably linked to a mammalian ubiquitin C promoter or a human CMV-IE promoter. 
     
     
       35. The isolated polynucleotide of  claim 34 , wherein the anti-AngPtl4 antibody light chain comprises the amino acid sequence of SEQ ID NO: 38. 
     
     
       36. The isolated polynucleotide of  claim 34 , wherein the anti-AngPtl4 antibody light chain comprises the amino acid sequence of SEQ ID NO: 37. 
     
     
       37. The isolated polynucleotide of  claim 34  comprising the nucleotide sequence of SEQ ID NO: 41. 
     
     
       38. The isolated polynucleotide of  claim 34  comprising the nucleotide sequence of SEQ ID NO: 42.

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