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CN-119432815-B - Fucosidase mutant and use thereof

CN119432815BCN 119432815 BCN119432815 BCN 119432815BCN-119432815-B

Abstract

The present disclosure relates to a novel mutant form of alpha-fucosidase (alpha-L-fucosidase) exhibiting enhanced alpha- (1, 6) fucosidase activity. The disclosure also relates to compositions comprising the novel mutant forms of alpha-fucosidase and methods of cleaving alpha- (1, 6) -linked fucose in glycoconjugates using the novel mutant forms of alpha-fucosidase.

Inventors

  • WU WEISHEN
  • ZHU GUOQING

Assignees

  • 醣基生医股份有限公司

Dates

Publication Date
20260508
Application Date
20240719
Priority Date
20230728

Claims (9)

  1. 1. A mutant alpha-L-fucosidase has an amino acid sequence shown in any one of SEQ ID NOs 2-18.
  2. 2. A method of preparing a defucosylated glycoconjugate in vitro, the method comprising sequentially contacting a glycoconjugate comprising one or more fucoses with an endoglycosidase and the mutant α -L-fucosidase according to claim 1.
  3. 3. The method according to claim 2, comprising the following sequential steps: (a) Contacting the glycoconjugate with the endoglycosidase, and (B) Contacting the glycoconjugate with the mutant α -L-fucosidase according to claim 1.
  4. 4. The method of claim 3, further comprising (c) terminating the reaction.
  5. 5. The method of claim 4, wherein step (C) is performed at 65 ℃ for 15-25 minutes.
  6. 6. The method of any one of claims 3 to 5, wherein the step (a) and/or the step (b) is performed at 37 ℃ for 0.5-2 hours.
  7. 7. The method according to any one of claims 3 to 5, wherein the endoglycosidase is endo- β -N-acetylglucosaminidase (NAG), endoA, endoF1, endoF2, endoF3, endoH, endoM, endoS, endoS2 and variants thereof.
  8. 8. The method of claim 7, wherein Endos2 comprises a substitution mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T M, T138N, T138Q, T138R, T V, T35138W, D182Q, D49226Q, T227Q and T228Q.
  9. 9. The method of any one of claims 2 to 5, wherein the fucose is an a- (1, 6) -linked fucose.

Description

Fucosidase mutant and use thereof Technical Field The present disclosure relates to a novel mutant form of alpha-fucosidase (alpha-L-fucosidase) exhibiting enhanced alpha- (1, 6) fucosidase activity. The disclosure also relates to compositions comprising the novel mutant forms of alpha-fucosidase and methods of cleaving alpha- (1, 6) -linked fucose in glycoconjugates using the novel mutant forms of alpha-fucosidase. Background N-glycosylation is one of the most common post-translational modifications observed in mammalian proteins and plays a vital role in regulating the intrinsic properties and biological functions of these proteins. For example, N-glycan attachment can significantly affect protein folding, stability, antigenicity, and immunogenicity. In addition, N-glycans can be directly involved in a wide range of biological recognition processes, including cell adhesion, host-pathogen interactions, cancer metastasis, and immune responses. Although mammalian N-glycans share a basic oligosaccharide core structure, the introduction of additional modifications to the core structure (such as sialylation and fucosylation) helps to increase the level of structural diversity that affects biological functions. The physiological activity of therapeutic antibodies is mediated by two different mechanisms. The first mechanism involves neutralization or apoptosis of the target antigen, which helps to improve therapeutic efficacy. The second mechanism involves antibody effector functions, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that are activated by formation of immune complexes. The significance of ADCC in the clinical efficacy of therapeutic antibodies, particularly anti-cancer antibodies, has been elucidated by genetic analysis of patient leukocyte receptor (fcγr) polymorphisms. Accordingly, the biological industry has been largely developing ADCC enhancement techniques comprising modification of N-glycans attached to antibody constant regions (fcs). The amount of alpha- (1, 6) -linked fucose (core fucose) attached to the innermost GlcNAc of the N-glycans attached to the Fc region of the antibody is known to affect ADCC activity. Removal of core fucose from the innermost GlcNAc of the N-glycans on the Fc region significantly enhances ADCC activity of IgG because of the increased binding affinity of non-fucosylated antibodies to fcyriii alpha receptors. Many approaches have been developed to enhance fcyriiiα binding and ADCC by reducing fucosylation of IgG. Some strategies involve the development of producer cell lines that eliminate or reduce the expression of alpha- (1, 6) fucosyltransferases. Alternative strategies to reduce fucosylation include silencing the α - (1, 6) fucosyltransferase gene by using RNAi. However, enzymatic defucosylation of N-glycans remains challenging in vitro because the N-glycans are embedded between two Fc domains. The presence of the Fc domain limits access of the α -fucosidase to the fucose residue, creating steric hindrance and resulting in less efficient enzymatic defucosylation. Only a few alpha-L-fucosidases, such as alpha-L-fucosidase from bovine kidney, alpha-L-fucosidase from human (FucA) and alpha-L-fucosidase from bacteroides fragilis (Bacteroides fragilis) (BfFuc) and alpha-L-fucosidase from lactobacillus casei (Lactobacilluscasei) C (AlfC), have been disclosed to be able to remove core fucose from the innermost GlcNAc residues after removal of the external N-glycans on the Fc region. Because of the limited and low inherent function and activity of wild-type enzymes, there is a need to develop a modified enzyme to cleave core fucose from N-glycans efficiently. Disclosure of Invention In one embodiment, the present disclosure provides a mutant form of α -L-fucosidase for use in improving enzymatic hydrolysis of fucose in vitro. In one embodiment, the present disclosure provides a mutant α -L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID No. 1 and a substitution mutation at amino acid position 247 (K247). In one embodiment, the present disclosure provides a mutant α -L-fucosidase comprising a polypeptide having at least 97% sequence identity to SEQ ID No. 1 and a substitution mutation at amino acid position 247 (K247). In one embodiment, the present disclosure provides a mutant α -L-fucosidase comprising a polypeptide having at least 99% sequence identity to SEQ ID No. 1 and a substitution mutation at amino acid position 247 (K247). In one embodiment, the present disclosure provides a mutant α -L-fucosidase comprising a polypeptide having the sequence of SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247). In some embodiments, the substitution mutation is selected from the group consisting of K247A, K C, K247D, K247E, K F, K247G, K247I, K247L, K247M, K247P, K247Q, K247S, K247T, K247V, K W and K247Y. In some embodiments, the mutant alpha-L-fucosidase has the s