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EP-3904388-B1 - FUCOSIDASE FROM BACTEROIDES AND METHODS USING THE SAME

EP3904388B1EP 3904388 B1EP3904388 B1EP 3904388B1EP-3904388-B1

Inventors

  • WONG, CHI-HUEY
  • TSAI, TSUNG-I

Dates

Publication Date
20260506
Application Date
20150527

Claims (4)

  1. A composition comprising an α-fucosidase, wherein the α-fucosidase comprises an isolated polypeptide having the sequence of SEQ ID NO: 1.
  2. The composition of claim 1, wherein the composition further comprises at least one glycosidase selected from the group consisting of Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM and EndoS.
  3. The composition of claim 1, wherein the α-fucosidase can hydrolyze α-(1,2), α-(1,3), α-(1,4), and α-(1,6)-linked fucoses present in N- and/or O- linked glycans in a glycoconjugate.
  4. The composition of claim 1, wherein the α-fucosidase has a pH optimum at 4-9.

Description

RELATED APPLICATIONS This application claims the benefit of US provisional aplication USSN 62/020,199, filed July 2, 2014. BACKGROUND OF THE INVENTION Fucose is an important component of many O- or N-linked oligosaccharide structures of glycoconjugates. Fucose-containing glycans are involved in numerous biological events, including development and apoptosis, and are involved in the pathology of inflammation, cancer, and cystic fibrosis. Defucosylation of the glycoconjugates is an important process for understanding the biological effects of the glycoconjugates. α-L-fucosidases (α-fucosidase) are exo-glycosidases, responsible for the removal of fucose residues from the non-reducing end of glycoconjugates by hydrolyzing α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages of fucoses attached, primarily to galactose or N-acetylglucosamine. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Antibody-dependent cellular cytotoxicity (ADCC) has been found to be one of the important effector functions responsible for the clinical efficacy of therapeutic antibodies. ADCC is triggered upon the binding of lymphocyte receptors (FccRs) to the antibody Fc region. ADCC activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-(1,6) linkage. US 7,090,973, and Database EMBL [Online] 3 March 2005, "Bacteroides fragilis NCTC 9343 putative exported alpha-L-fucosidase protein" disclose the bacterial genome of Bacteroides fragilis NCTC 9343. Database EMBL [Online] 6 January 2006, "Bacteroides thetaiotaomicron VPI-5482 alpha-L-fucosidase precursor" discloses the bacterial genome of Bacteroides thetaiotaomicron. Shih-Fen Liao et al. "Immunization of fucose-containing polysaccharides from Reishimushroom induces antibodies to tumor-associated Globo H-series epitopes" PNAS vol. 110, no. 34, 1 August 2013, pages 13809-13814 discloses the enzymatic hydrolysis of alpha-linked fucose residues from fucose-enriched Reishi polysaccharide fraction using alpha-fucosidase derived from Bacteroides fragilis. Haruko Sakurama et al. "Differences in the substrate specificities and active-site structures of two [alpha]-L-fucosidases (Glycoside Hydrolasae Family 29) from Bacteroides thetaiotaomicron" Bioscience Biotechnology Biochemistry, vol. 76, no. 5, 23 May 2012, pages 1022-1024 discloses two alpha-fucosidases derived from Bacteroides thetaiotaomicron in methods for testing their substrate specificity. Wei Huang et al. "Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions" Journal of the American Chemical Society, vol. 134, no. 29, 25 July 2012, pages 12308-12318 discloses the use of an alpha-fucosidase from Bacteroides fragilis in the in vitro enzymatic defucosylation of glycoproteins. WO 2013/120066 discloses the use of an alpha-fucosidase from Bacteroides fragilis in the in vitro enzymatic defucosylation of glycoproteins. SUMMARY OF THE INVENTION Accordingly, the present invention provides the compositions and methods for the improved enzymatic hydrolysis of fucose in vitro. In particular, the present invention is useful for the efficient cleavage of core fucose in native glycoproteins without denaturation or functional deterioration of glycoproteins. The compositions and methods of the invention can facilitate the Fc glycoengineering of Fc fusion proteins or antibodies, such as therapeutic antibodies. This invention also provides the application of glycan sequencing for distinguishing fucose position on a glycoconjugate. The glycoconjugate may be a glycolipid, glycoprotein, oligosaccharide, or glycopeptide. The invention provides a composition comprising an α-fucosidase, wherein the α-fucosidase comprises an isolated polypeptide having the sequence of SEQ ID NO: 1. In some embodiments, the composition further comprises at least one glycosidase selected from the group consisting of Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM and EndoS. In some embodiments, the α-fucosidase can hydrolyze α-(1,2), α-(1,3), α-(1,4), and α-(1,6)- fucoses present in N- and/or O- linked glycans in a glycoconjugate. In some embodiments, the α-fucosidase has a pH optimum at 4-9. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. shows the biochemical properties of BfFucH.Figure 2. (a) pH profile of BfFucH (b) temperature effects on the enzyme activity of BfFucH (c) metal ion effects on the enzyme activity of BfFucH.Figure 3. shows the time course of of BfFucH treatment for Rituxan. DETAILED DESCRIPTION OF THE INVENTION The absence of core fucose residues in the Fc glycans is known to substantially increase the ADCC activity of IgG as nonfucosylated antibodies bind to the FcgRIIIα receptor with significantly increased affinity. To improve FcgRIIIα binding and ADCC, several strategies have been developed to reduce fucosylation of IgG, including the development of production cell lines that abolish or reduce expressio