Method of producing antibodies with modified fucosylation level

A technology of fucosylation and fucose, applied in chemical instruments and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of impractical therapeutic antibody products and low antibody production

Inactive Publication Date: 2008-08-06
GENENTECH INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, lectin mutants of the YB2 / 0 (rat myeloma) and Lec13 (CHO lines) have defective GDP-mannose 4,6-dehydratase, resulting in a substrate for α1,6-fucosyltransferase GDP-fucose or GDP-sugar intermediate deficient (Ripka et al., 1986)) cell lines can produce antibodies with 78-98% afucosylated type
Unfortunately, antibody yields from these cells are extremely low, making these cell lines impractical for commercial production of therapeutic antibody products

Method used

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  • Method of producing antibodies with modified fucosylation level
  • Method of producing antibodies with modified fucosylation level
  • Method of producing antibodies with modified fucosylation level

Examples

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Embodiment 1

[0401] Example 1: Conversion of Existing Cell Lines to Less Fucosylated Cell Lines

[0402]To achieve high yields of afucosylated antibodies in CHO cells, RNAi was used to knock down the expression of the FUT8 gene. The pSilencer3.1-H1-Puro plasmid from Ambion Corporation (Austin, TX) was used to generate short hairpin siRNA consisting of a 19 nt (nucleotide) sense siRNA sequence specific to the FUT8 gene and its reverse complementary antisense The siRNA sequences are joined by a short spacer (9nt hairpin loop) followed by 5-6 U's at the 3' end (Figure 3). For the method for designing siRNA probes targeting CHO FUT8 gene, see Elbashir et al., 2002. Five different siRNA probes (probes #1-5) were designed to target different regions based on the available CHO FUT8 DNA sequence (Figure 4). Probe 1 (SEQ ID NO.3 and NO.4); Probe 2 (SEQ ID NO.5 and NO.6); Probe 3 (SEQ ID NO.7 and NO.37); Probe 4 (SEQ ID NO.38 and NO.39); Probe 5 (SEQ ID NO.40 and NO.41). The siRNA coding sequenc...

Embodiment 2

[0406] Example 2: Manipulation of fucose content of stably expressed antibodies by transient siRNA expression

[0407] The RNAi2 and RNAi4 plasmids were transiently transfected into a previously established stable CHO cell line (clone #60) expressing the humanized anti-CD20 antibody 2H7.v16. Transfected cells were then inoculated separately into 250 ml spinner flasks in serum-free medium for antibody production.

[0408] The 2H7.v16 antibody expressed and secreted in the harvested cell culture fluid was purified with protein A columns and analyzed by matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF) as described in Papac et al., 1998N - Fucose content of linker oligosaccharides. Antibodies were also assayed in an Fc[gamma]R binding assay (described below). There are three classes of human Fcy receptors: FcyRI, FcyRII and FcyRIII. Some of these have functional allelic polymorphisms, giving rise to allotypes with different receptor prope...

Embodiment 3

[0417] In this example, we constructed a novel RNAi plasmid containing two RNAi transcription units targeting two different regions of the FUT8 gene. This plasmid is more effective than previous plasmid types that target only one region of the gene.

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Abstract

The present invention provides methods for controlling the level of fucosylation and enhancing ADCC activity in antibodies by inhibiting FUT8 expression in antibody-producing cells.

Description

[0001] This application claims the benefit of US Provisional Application Serial Nos. 60 / 687,625, filed June 3, 2005, and 60 / 736,982, filed November 14, 2005, the entire disclosures of which are incorporated herein by reference. field of invention [0002] The present invention relates to the production of antibodies with reduced fucose and improved Fc function. Background of the invention [0003] Recombinant therapeutic proteins are routinely produced in several mammalian host cell lines, including murine myeloma NSO and Chinese hamster ovary (CHO) cells (Anderson and Krummen, 2002; Chu and Robinson, 2001). Each cell line has its advantages and disadvantages in terms of productivity and the identity of the proteins produced by the cell. The selection of commercial production cell lines often balances the need for high productivity with the ability to present the desired product quality characteristics for a given product. An important class of therapeutic recombinant prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/28C12N9/10C12N15/11C12N15/13A61K39/395C12N5/10C12N15/113C12N15/85
Inventor 约翰·C·乔利亨利·B·洛曼多明戈斯·恩格埃米·Y·申布拉德利·R·斯内德科尔
Owner GENENTECH INC
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