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Fusion protein for remodeling antibody glycoform

A fusion protein and antibody technology, applied in the direction of fusion polypeptide, antibody mimic/scaffold, application, etc., can solve problems such as low efficiency

Pending Publication Date: 2022-02-08
CHO PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, existing fucosidases are inefficient at cleaving core fucose from the Fc region

Method used

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  • Fusion protein for remodeling antibody glycoform
  • Fusion protein for remodeling antibody glycoform
  • Fusion protein for remodeling antibody glycoform

Examples

Experimental program
Comparison scheme
Effect test

example

[0089] fusion protein

example 1

[0090] Example 1: Production of Fusion Proteins

[0091] Generation of full-length or truncated (IgG binding motif only) containing fusions to fucosidases BF3242 (Table 1), Alfc (Table 2), BT2970 (Table 3), EO0918 (Table 4) and Emfuc3 (Table 5) Fusion proteins of EndoS or EndoS2, as shown in Tables 1 to 5 below.

[0092] Table 1. BF3242 fusion protein

[0093]

[0094]

[0095] a Only the IgG binding domain of endoS or endoS2

[0096] b wild-type endoS or endoS2

[0097] Table 2. Alfc fusion proteins

[0098]

[0099]

[0100] Table 3. BT2970 fusion proteins

[0101]

[0102]

[0103] Table 4. EO0918 fusion protein

[0104]

[0105] Table 5. Emfuc3 fusion proteins

[0106]

[0107] Remodeling of glycans in antibody Fc regions.

example 2

[0108] Example 2: Analysis of fucosidase activity

[0109] The test antibody (ie, Trastuzumab (TRZ)) was treated with endoS2 in Tris-HCl (pH 7.0) at 37°C for 1 hour. The treated antibody was adsorbed on protein A affinity chromatography resin (MabSelect TM ), washed with Tris-HCl (pH 7.4) and eluted with citrate buffer (pH 3.0) for purification. The majority of residual glycans in the Fc region of the resulting antibody are N-acetylglucosamine-fucose (GlcNAc-Fuc). Purified antibodies were treated with fusion protein or natural fucosidase in Tris-HCl buffer (pH 7.0) at 37°C for 16h, followed by heating at 55°C for 20min to inactivate the enzyme activity. The reaction mixture was filtered through a 0.22 μm membrane and the treated antibody was purified by affinity chromatography.

[0110] The content of residual fucose was determined by analyzing the mass of intact protein. TRZ-N / N (TRZ containing two GlcNAc in the Fc region), TRZ-N / NF (TRZ containing one GlcNAc and one GlcN...

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Abstract

The present disclosure provides a fusion protein comprising a fucosidase or a truncated fragment or a mutant thereof fuses with either N-terminal end or C-terminal end of the endoglycosidase or a truncated fragment of mutant thereof. The present disclosure also provides a nucleic acid molecule expressing the fusion protein and a method for remodeling a glycan of an antibody Fc region.

Description

technical field [0001] The present invention relates to glycoform remodeling; in particular, to fusion proteins for remodeling antibody glycoforms. Background technique [0002] Antibodies, also known as immunoglobulins (Ig), are glycoproteins that play a major role in the immune response. IgG antibodies and fragments of IgG antibodies have been developed as important biotherapies for the treatment of a variety of diseases. The constant (Fc) region of native antibodies is usually N-glycosylated. Specific glycosylation (ie, glycoforms) significantly affects Fc effector function and antibody stability. Thus, one approach to improving the therapeutic efficacy of antibodies is to modify the glycoforms in the Fc region. [0003] Fc glycoforms typically include a core fucose residue. In the context of cancer therapy, anti-tumor antibodies with glycoforms in the Fc region and lower core fucose content have been reported to display higher binding affinity to CD16 (FcγRIIIA) on N...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56
CPCC12N9/2402C12Y302/01127C12Y302/01051C07K2319/00C12N15/52C12N15/62
Inventor 朱国庆黄琳雅曾意芳
Owner CHO PHARMA INC
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