Glycosyl transferase from Chinese hamster and related methods

• US 8,609,372 B2
• Filed: 11/10/2010
• Issued: 12/17/2013
• Est. Priority Date: 11/11/2009
• Status: Active Grant

• Current Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Original Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Inventors: Meador, James W. III
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US12/943,719
• Publication Number:
  US 20110275526A1
• Time in Patent Office: 1,133 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

A glycosyl transferase from Chinese hamster and related methods are described.

Claims

1. A cDNA molecule comprising

(i) a nucleic acid having at least 95% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2, wherein the nucleic acid encodes a polypeptide that catalyzes synthesis of a Gal-α

1,3-Gal glycan;

(ii) a nucleic acid having at least 96% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

4, wherein the nucleic acid encodes a polypeptide that catalyzes synthesis of Gal-α

1,3-Gal glycan;

(iii) a nucleic acid having at least 99% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

7, wherein the nucleic acid encodes a polypeptide that catalyzes Gal-α

1,3-Gal glycan;

or

(iv) the complement of the nucleic acid of (i), (ii), or (iii).

2. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 96% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2, or (vi) the complement of the nucleic acid of (v).

3. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 97% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2, or (vi) the complement of the nucleic acid of (v).

4. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 98% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2, or (vi) the complement of the nucleic acid of (v).

5. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 99% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2, or (vi) the complement of the nucleic acid of (v).

6. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 97% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

4, or (vi) the complement of the nucleic acid of (v).

7. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 98% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

4, or (vi) the complement of the nucleic acid of (v).

8. The cDNA molecule of claim 1, wherein the cDNA molecule comprises (v) a nucleic acid having at least 99% identity to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

4, or (vi) the complement of the nucleic acid of (v).

13. A nucleic acid construct comprising the sequence of the cDNA molecule of claim 1.

14. A vector comprising the cDNA molecule of claim 1.

9. A cDNA molecule encoding the sequence of SEQ ID NO:

2, or SEQ ID NO;

4 or SEQ ID NO;

7.

10. A cDNA molecule having at least 90% sequence identity to the sequence of SEQ ID NO:

1 or SEQ ID NO;

3 or SEQ ID NO;

6, wherein the cDNA encodes a polypeptide that catalyzes synthesis of a Gal-α

1,3-Gal glycan.

12. A nucleic acid molecule comprising the cDNA molecule of any one of claims 10 and 11, linked in-frame to a nucleic acid encoding a heterologous amino acid sequence.

15. A two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, wherein at least one address of the plurality has a capture probe comprising a cDNA molecule of claim 9, 10, or 11.

16. An isolated host cell transfected with an expression vector comprising the cDNA molecule of any one of claims 10 and 11.

17. The isolated host cell of claim 16, wherein the host cell produces a recombinant therapeutic glycoprotein having increased levels of terminal galactose-α

1,3-galactose glycans compared to the recombinant therapeutic glycoprotein produced by parent cells of the host cell.

18. The isolated host cell of claim 17, wherein the cell is a Chinese hamster ovary (CHO) cell engineered to express a recombinant therapeutic glycoprotein.

19. The isolated host cell of claim 16, wherein the host cell produces a recombinant therapeutic glycoprotein having lower levels of terminal galactose-α

1,3-galactose glycans compared to the recombinant therapeutic glycoprotein produced by parent cells of the host cell.

20. The isolated host cell of claim 19, wherein the cell is a CHO cell engineered to express a recombinant therapeutic glycoprotein.

21. A method of modulating the glycan structure of a recombinant therapeutic glycoprotein produced in a CHO cell, the method comprising culturing the isolated host cell of claim 18 under conditions sufficient to express the recombinant therapeutic glycoprotein.

22. A method of modulating the glycan structure of a recombinant therapeutic glycoprotein produced in a CHO cell, the method comprising culturing the isolated host cell of claim 20 under conditions sufficient to express the recombinant therapeutic glycoprotein.

23. A method of detecting Ggta1 expression in a CHO cell population, the method comprising:

hybridizing a nucleic acid sample from the CHO cell population with the cDNA molecule of claim 9, 10, or 11, wherein the cDNA molecule further comprises a label, to thereby detect Ggta1 expression in the CHO cell population.

24. A method of detecting Ggta1 expression in a CHO cell population, the method comprising obtaining a nucleic acid sample from the CHO cell population, amplifying a Ggta1 nucleic acid sequence in the nucleic acid sample using PCR, and detecting the amplified Ggta1 molecules with the cDNA molecule of claim 9, 10, or 11, wherein the cDNA molecule further comprises a label.

25. The method of claim 23, further comprising quantifying the level of Ggta1 expression in the CHO cell population.

26. The method of claim 25, further comprising comparing the level of Ggta1 expression in the CHO cell population to a reference level, a control level, a pre-determined level or a level exhibited by a second CHO cell population.

27. A method of analyzing a nucleic acid sample from a CHO cell, the method comprising contacting the nucleic acid sample to the array of claim 15.

11. A cDNA molecule comprising the nucleotide sequence of SEQ ID NO:

1 or SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

6, or a complement thereof.