KR-20260066105-A - DNASE co-expression in host cells

Abstract

The present disclosure relates to the introduction into a host cell line at a defined ratio comprising a plurality of dock sites for the insertion of a nucleic acid construct of an expression construct encoding a different gene product, such as a protein or nucleic acid, and to the production of a protein requiring the expression of at least two gene products.

Inventors

• 스퀸토, 스티븐
• 루드비히, 데일
• 키하노, 엘리아스
• 글레이저, 피터
• 블렉, 그레고리

Assignees

• 예일 유니버시티

Dates

Publication Date

20260512

Application Date

20240830

Priority Date

20230901

Claims (20)

1. A eukaryotic host cell comprising (i) an exogenous nucleic acid sequence encoding an endonuclease operably linked to a first promoter sequence and (ii) an exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a second promoter sequence.
2. In claim 1, the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 cell line transformed by SV40, baby hamster kidney cells, mouse Sertori cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, dog kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human liver cancer cell line cells.
3. In claim 1, the host cell is a host cell selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, and CAP cells.
4. In claim 1 or 2, the host cell line is a glutamine synthase (GS) knockout cell line.
5. In any one of claims 1 to 3, the host cell line is a dihydrofolate reductase (DHFR) knockout cell line.
6. In any one of claims 1 to 5, the host cell, wherein the endonuclease is a DNAse I endonuclease.
7. In paragraph 6, the above endonuclease is a human DNAse I endonuclease, a host cell.
8. In any one of claims 1 to 5, the host cell, wherein the endonuclease is a DNAse II endonuclease.
9. In paragraph 8, the above endonuclease is a human DNAse II endonuclease, a host cell.
10. In any one of claims 1 to 9, the host cell, wherein the sequence encoding the endonuclease is additionally operably linked to a secretion signal sequence.
11. In any one of claims 1 to 10, the host cell, wherein the first promoter sequence is a weak promoter sequence.
12. In any one of claims 1 to 10, the host cell, wherein the first promoter sequence is a cytomegalovirus immediate early (CMV-IE) promoter sequence.
13. In paragraph 12, the above CMV-IE promoter sequence is a host cell, which is an ape CMV-IE promoter (sCMV) sequence.
14. In any one of claims 1 to 11, the first promoter sequence is a host cell, not a retrovirus LTR promoter.
15. In any one of paragraphs 1 through 14, The above 3E10 antibody or its antigen-binding fragment comprises (a) a heavy chain polypeptide or a fragment thereof and (b) a light chain polypeptide or a fragment thereof; The exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment comprises (1) a first exogenous nucleic acid sequence encoding the heavy chain polypeptide or its fragment and (2) a second exogenous nucleic acid sequence encoding the light chain polypeptide or its fragment, in a host cell.
16. In paragraph 15, a host cell in which the ratio of (i) the number of first exogenous nucleic acid sequences incorporated into the host cell genome to (ii) the number of second exogenous nucleic acid sequences incorporated into the host cell genome is 1:2 to 2:1.
17. A host cell according to claim 15 or 16, wherein the first exogenous nucleic acid sequence is operably linked to the second promoter sequence and the second exogenous nucleic acid sequence is operably linked to the third promoter sequence.
18. In paragraph 17, the third promoter sequence is a host cell having the same nucleotide sequence as the first promoter.
19. In claim 17 or 18, the host cell, wherein the third promoter sequence has the same nucleotide sequence as the second promoter.
20. In paragraph 17, the host cell, wherein the third promoter sequence has a nucleotide sequence different from the first promoter.

Description

DNASE co-expression in host cells Cross-reference regarding related applications This application claims priority to U.S. provisional patent application no. 63/580,228 filed September 1, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Statement regarding government-supported investigation This invention was made with government support pursuant to CA197574 of the National Institutes of Health. The government holds specific rights to this invention. The present disclosure provides a host cell that allows co-expression of an endonuclease and a product of interest, e.g., a 3E10 antibody or its antigen-binding fragment, thereby improving the expression and/or purification of a product of interest, e.g., a 3E10 antibody or its antigen-binding fragment, from a host cell culture. Therapeutic recombinant products are widely used for human medical purposes. Recombinant proteins can be expressed in various types of organisms. CHO and HEK cells are some of the most commonly used host cells for the manufacture of biopharmaceuticals. While using host cells for the production of therapeutics offers many advantages, contamination of biopharmaceuticals by host cell DNA is a major concern for manufacturers. Genomic DNA contamination in biopharmaceuticals has been considered a potential risk factor for patients receiving recombinant protein drugs. Therefore, biopharmaceutical manufacturers need to monitor DNA contamination and keep the amount of impurities in biological products as low as the safety limits set by regulatory authorities such as the FDA, which generally need to be lower than 100 pg of DNA per milligram of protein. Removing unwanted DNA is costly, time-consuming, and can reduce the recovery efficiency of biopharmaceutical proteins. Therefore, an approach to limit contamination of biopharmaceutical protein preparations by host cell DNA is needed in this technical field. The present disclosure provides a host cell that allows co-expression of DNAse and a product of interest, e.g., a 3E10 antibody or an antigen-binding fragment thereof, thereby improving the expression and/or purification of a protein of interest from a host cell culture. In some embodiments, the present disclosure provides a eukaryotic host cell comprising (i) an exogenous nucleic acid sequence encoding an endonuclease operably linked to a first promoter sequence and (ii) an exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a second promoter sequence. In some embodiments, the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 cell line transformed by SV40, baby hamster kidney cells, mouse Sertori cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, dog kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human liver cancer cell line cells. In some embodiments, the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, and CAP cells. In some embodiments, the host cell line is a glutamine synthase (GS) knockout cell line. In some embodiments, the host cell line is a dihydrofolate reductase (DHFR) knockout cell line. In some embodiments, the endonuclease is a DNAse I endonuclease. In some embodiments, the endonuclease is human DNAse I endonuclease. In some embodiments, the sequence encoding the endonuclease is additionally operably linked to the secretion signal sequence. In some embodiments, the first promoter sequence is a weak promoter sequence. In some embodiments, the first promoter sequence is the cytomegalovirus immediate early (CMV-IE) promoter sequence. In some embodiments, the CMV-IE promoter sequence is the primate CMV-IE promoter (sCMV) sequence. In some embodiments, the first promoter sequence is not a retrovirus LTR promoter sequence. In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises (a) a heavy chain polypeptide or a fragment thereof and (b) a light chain polypeptide or a fragment thereof, and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment comprises (1) a first exogenous nucleic acid sequence encoding a heavy chain polypeptide or a fragment thereof and (2) a second exogenous nucleic acid sequence encoding a heavy chain polypeptide or a fragment thereof. In some embodiments, the ratio of (i) the number of first exogenous nucleic acid sequences incorporated into the host cell genome to (ii) the number of second exogenous nucleic acid sequences incorporated into the host cell genome is 1:2 to 2:1. In some embodiments, the first exogenous nucleic acid sequence is operably linked to the second promoter sequence, and the second exogeno