Claims for Patent: 7,807,804

Summary for Patent: 7,807,804

Title:	Methods and compositions for improving recombinant protein production
Abstract:	Nucleic acid molecules modified to enhance recombinant protein, e.g., antibody, expression and/or reduce or eliminate mis-spliced and/or intron read-through (IRT) by-products are disclosed. The invention also provides methods for producing proteins devoid of mis-spliced and/or intron read-through by-products by the use of such vectors in host cells under cell culture conditions suitable for recombinant protein expression.
Inventor(s):	Sinacore; Martin S. (Andover, MA), Leonard; Mark (Manchester, NH), Laken; Haley A. (Pepperell, MA), Rouse; Jason (Londonderry, NH)
Assignee:	Wyeth LLC (Madison, NJ)
Application Number:	11/244,678
Patent Claims:	1. A purified nucleic acid molecule comprising a nucleotide sequence having one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally-occurring genomic sequence and wherein the exon sequence comprises one or more of a second constant region (C.sub.H2) exon or a third constant region (C.sub.H3) exon, thereby reducing a mis-spliced or an intron read-through (IRT) by-product, and wherein said nucleotide sequence encodes an antibody heavy chain or a fragment thereof. 2. A purified nucleic acid molecule comprising a nucleotide sequence comprising one or more intron and exon sequences, wherein at least three intron sequences are deleted compared to the naturally-occurring genomic sequence and wherein the exon sequence comprises one or more of a second constant region (C.sub.H2) exon or a third constant region (C.sub.H3) exon, thereby enhancing protein expression, and wherein said nucleotide sequence encodes an antibody heavy chain or a fragment thereof. 3. The purified nucleic acid molecule of claim 1, wherein the antibody heavy chain or fragment thereof comprises one or more of a heavy chain variable region, a hinge region, a first constant region (C.sub.H1), a second constant region (C.sub.H2), or a third constant region (C.sub.H3) of a human immunoglobulin G subtype, or a mutated form thereof. 4. The purified nucleic acid molecule of claim 2, wherein the antibody heavy chain or fragment thereof comprises one or more of a heavy chain variable region, a first constant region (C.sub.H1), a second constant region (C.sub.H2), or a third constant region (C.sub.H3) of a human immunoglobulin G subtype, or a mutated form thereof. 5. The purified nucleic acid molecule of claim 3, wherein the immunoglobulin G subtype is a human IgG1 or human IgG4, or a mutated form thereof. 6. The purified nucleic acid molecule of claim 4, wherein the immunoglobulin G subtype is a human IgG1 or human IgG4, or a mutated form thereof. 7. The purified nucleic acid molecule of claim 3, wherein an intron between the CH2 region and the CH3 region of the immunoglobulin heavy chain constant region is deleted. 8. The purified nucleic acid molecule of claim 4, wherein an intron between the C.sub.H1 region and the hinge region, an intron between the hinge region and the C.sub.H2 region, and an intron between the C.sub.H2 region and the C.sub.H3 region of the immunoglobulin heavy chain constant region are deleted. 9. The purified nucleic acid molecule of claim 3, wherein the nucleic sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence at least 95% identical to the nucleotide sequence shown in FIG. 8 (SEQ ID NO:1), or at least 95% identical to the nucleotide sequence shown in FIG. 9 (SEQ ID NO:3). 10. The purified nucleic acid molecule of claim 4, wherein the nucleic sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence at least 95% identical to the nucleotide sequence shown in FIG. 8 (SEQ ID NO:1), or at least 95% identical to the nucleotide sequence shown in FIG. 9 (SEQ ID NO:3). 11. The purified nucleic acid molecule of claim 7, wherein the deletion of the intron between C.sub.H2 and C.sub.H3 corresponds to nucleotides between 1409 to 1505 of human IgG1 as shown in FIG. 8 (SEQ ID NO:1), or about nucleotides 1401 to 1497 of human IgG4 as shown in FIG. 9 (SEQ ID NO:3). 12. The purified nucleic acid molecule of claim 8, wherein the deletion of the intron between C.sub.H2 and C.sub.H3 corresponds to nucleotides between 1409 to 1505 of human IgG1 as shown in FIG. 8 (SEQ ID NO:1), or about nucleotides 1401 to 1497 of human IgG4 as shown in FIG. 9 (SEQ ID NO:3). 13. The purified nucleic acid molecule of claim 8, wherein the deletion of the intron between C.sub.H1 and the hinge region corresponds to about nucleotides 525 to 915 of human IgG1 as shown in FIG. 8 (SEQ ID NO:1), or about nucleotides 525 to 916 of human IgG4 as shown in FIG. 9 (SEQ ID NO:3). 14. The purified nucleic acid molecule of claim 8, wherein the deletion of the intron between the hinge region and C.sub.H2 corresponds to about nucleotides 961 to 1078 of human IgG1 as shown in FIG. 8 (SEQ ID NO:1), or about nucleotides 953 to 1070 of human IgG4 as shown in FIG. 9 (SEQ ID NO:3). 15. A purified nucleic acid molecule comprising a nucleotide sequence encoding human IgG1, wherein said nucleotide sequence is at least 90% identical to the sequence shown in FIG. 10 (SEQ ID NO:5), or at least 90% identical to the sequence shown in FIG. 11 (SEQ ID NO:6). 16. A purified genomic nucleotide sequence encoding a human IgG1 or IgG4 heavy chain constant region, or a mutated form thereof, wherein said nucleotide sequence lacks at least one intron present in the naturally-occurring genomic sequence, and wherein said intron facilitates intron-read through. 17. A purified nucleic acid molecule comprising a nucleotide sequence represented by the formula: V.sub.H-Int1-C.sub.H1-Int2-Hinge-Int3-C.sub.H2-C.sub.H3, wherein V.sub.H is a nucleotide sequence encoding a heavy chain variable region; C.sub.H1, C.sub.H2, and C.sub.H3 are nucleotide sequences encoding a heavy chain constant region, wherein the heavy chain constant region is a human immunoglobulin G heavy chain constant region or a mutated form thereof; Hinge is a nucleotide sequence encoding hinge region of the heavy chain constant region; and Int1, Int2, and Int3 are introns from a heavy chain genomic sequence. 18. A purified nucleic acid molecule comprising a nucleotide sequence represented by the formula: V.sub.H-Int1-C.sub.H1-Hinge-C.sub.H2-C.sub.H3, wherein V.sub.H is a nucleotide sequence encoding a heavy chain variable region; C.sub.H1, C.sub.H2, and C.sub.H3 are nucleotide sequences encoding the corresponding heavy chain constant region, wherein the heavy chain constant region is a human immunoglobulin G heavy chain constant region or a mutated form thereof; Hinge is a nucleotide sequence encoding hinge region of the heavy chain constant region; and Int1 is an intron from a heavy chain genomic sequence. 19. An expression vector comprising the nucleic acid molecule of claim 1 or 3. 20. An expression vector comprising the nucleic acid molecule of claim 2 or 4. 21. A cultured host cell comprising the expression vector of claim 19. 22. A cultured host cell comprising the expression vector of claim 20. 23. A method of expressing, in a mammalian host cell, a recombinant antibody or fragment thereof substantially free of an intron read-through (IRT) product, comprising; identifying an IRT product in a nucleic acid sample from the host cell; introducing the purified nucleic acid molecule of claim 3 into the host cell; culturing said host cell under conditions that allow expression of the recombinant antibody or fragment thereof, thereby producing a culture of host cells; and obtaining the recombinant antibody or fragment thereof from the culture of host cells. 24. A method for enhancing expression of a recombinant antibody or fragment thereof, comprising: introducing the purified nucleic acid molecule of claim 4, and a nucleotide sequence encoding a light chain variable region and a constant region into a mammalian host cell; culturing said host cell under conditions that allow expression of the recombinant antibody, thereby producing a culture of host cells; and obtaining the recombinant antibody from the culture of host cells. 25. A method for producing a recombinant antibody or fragment thereof substantially devoid of intron read-through (IRT) heavy chain by-product, comprising: culturing a mammalian host cell comprising the purified nucleic acid molecule of claim 3 and a nucleic acid encoding an antibody light chain, under conditions such that the heavy and light chains are expressed. 26. The purified nucleic acid molecule of claim 1 or 2, wherein the antibody heavy chain or fragment thereof encoded by the purified nucleic acid expressed in a CHO cell line at least two fold higher than the expression of the antibody heavy chain or fragment thereof encoded by the naturally-occurring genomic sequence. 27. The purified nucleic acid molecule of claim 26, wherein the antibody heavy chain or fragment thereof comprises one or more of a heavy chain variable region, a hinge region, a first constant region (C.sub.H1), a second constant region (C.sub.H2), or a third constant region (C.sub.H3) of an immunoglobulin. 28. The purified nucleic acid molecule of claim 27, wherein the immunoglobulin is selected from the group consisting of IgG1, IgG3, and IgG4. 29. The purified nucleic acid molecule of claim 3 or 4, wherein the antibody heavy chain or fragment thereof comprises the heavy chain variable region, the hinge region, the first constant region (C.sub.H1), the second constant region (C.sub.H2), and the third constant region (C.sub.H3) of the human immunoglobulin, or mutated form thereof. 30. The purified nucleic acid molecule of claim 3 or 4, wherein the mutated form of the antibody heavy chain or fragment thereof results in one or more of: increased stability, reduced effector function, reduced complement fixation, reduced glycosylation, relative to the naturally-occurring form. 31. The purified nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule is modified by one or more of: rearranging the intron/exon configuration; deleting a portion of one or more introns; or replacing an intron or portion thereof with a heterologous intron sequence, such that when expressed enhanced protein expression and/or reduction or elimination of mis-spliced or intron read-through (IRT) by product occurs. 32. The purified nucleic acid molecule of claim 1 or 2, wherein the exon sequences comprise a heavy chain variable region exon, a hinge region exon, a first constant region (C.sub.H1) exon, a second constant region (C.sub.H2) exon, and a third constant region (C.sub.H3) exon of a human immunoglobulin G subtype, or a mutated form thereof. 33. The purified nucleic acid molecule of claim 1 or 2, wherein the intron sequence between the C.sub.H2 and C.sub.H3 exons is deleted. 34. The purified nucleic acid molecule of claim 1 or 2, further comprising one or more deletions of the intron sequences between wherein C.sub.H1and the hinge region, or between the hinge region and C.sub.H2. 35. A cultured host cell comprising the nucleic acid molecule of claim 1 or 2, wherein the host cell is transiently or stably transfected with said nucleic acid molecule. 36. The host cell of claim 35, wherein the host cell is cultured in large-scale. 37. The host cell of claim 35, wherein the host cell is a lymphocytic cell line or a COS cell line. 38. The host cell of claim 37, wherein the host cell is a COS cell line.

Details for Patent 7,807,804

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Csl Behring Ag	CARIMUNE, CARIMUNE NF, PANGLOBULIN, SANDOGLOBULIN	immune globulin intravenous (human)	For Injection	102367	07/27/2000		2024-10-05
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2024-10-05
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2024-10-05
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2024-10-05
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	07/26/2007		2024-10-05
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	10/02/2009		2024-10-05
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	02/07/2013		2024-10-05