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Engineered and fully-functional customized glycoproteins

A target protein, galactosyl technology, applied in the field of engineered and fully-functionally customized glycoproteins, which can solve problems such as reduced in vivo efficacy

Pending Publication Date: 2020-05-05
LIMMATECH BIOLOGICS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, the absence of terminal sialic acid on many therapeutic glycoproteins can reduce in vivo efficacy and thus require more frequent patient dosing regimens

Method used

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  • Engineered and fully-functional customized glycoproteins
  • Engineered and fully-functional customized glycoproteins
  • Engineered and fully-functional customized glycoproteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0433] 8.2 Example 2 Method for Glycoengineering Fully Functional-Customized N-Glycan Variants

[0434] Figure 7 with 8 Glycoengineering in lizard Leishmania (L. tarentolae) to generate uniform and functionally-tailored N-glycan extensions for a) desired Fc and Fc receptor interactions is documented Downstream effects and b) increased half-life (enhanced pharmacokinetic PK) and c) anti-inflammatory properties such as immune-tolerance benefits of 2,6-linked Neu5Ac to avoid ADA in potentially immunogenic protein therapeutics. Figure 8 Proposals for N-glycan biosynthesis in lizard Leishmania (L. tarentolae) showing major and novel differences leading to beneficial properties for glycoengineering are described. The present invention relates to eukaryotic host cells that have been modified to produce function-tailored proteins through heterologous expression of a group of glycosyltransferases, including N-acetylglucosyltransferase, galactosyltransferase, and sialyltransferase ...

Embodiment 3

[0435] 8.3 Example 3 Experimental Evidence for Reduced and 100% Uniform N-Glycans in the Kinetoplastid Lizard Leishmania tarentolae

[0436]Three wild-type (wt) strains (St10569, St10616, St11262) were analyzed for their native N-glycans after selection of L. tarentolae kinetoplastid organisms based on expected beneficial properties. First, cell pellets are denatured and subjected to PNGaseA or PNGaseF-mediated N-glycan release. By WatersGlycoWorks for fast enzymatic release and rapid labeling of N-glycans TM RapiFluor-MS TM N-Glycan either a) permethylates or b) marks the released N-glycans.

[0437] By using RapiFluor TM Fluorescence labeling and UPLC separation of , we only observed corresponding to (Man) 3 (GlcNAc) 2 A single N-glycan form of , calculated for m / z=1222.7166; [M+Na] + 1244.6985; [M+K] + 1260.6724. As for N-glycans, only one homogeneous N-glycan, the so-called oligomannose or Man3 glycan, was identified in all three isolated lizard Leishmania (L. ta...

Embodiment 8

[0471] 8.8 Example 8 Targeting and retention to the correct subcellular compartment

[0472] Such as Image 6 with 7 As listed, sequential processing of N-glycans by glycosyltransferases occurs in the endoplasmic reticulum (ER) and Golgi apparatus (kelokumpu et al., 2016). It is generally believed that the enzymes involved (glycosyltransferases and glycosidases) act individually and sequentially at a certain time, in the order indicated, by traveling to and from the growing oligosaccharide chain to add or remove sugar residues. This suggests that Gnt for glycoengineering can be improved by proper localization and retention along the secretory pathway. Most Golgi-localized Gnts are type II membrane proteins with a short N-terminal cytoplasmic domain ("C"), an a-helical TM domain ("T") of about 20 amino acids, a stem domain ("S") , and then the C-terminal globular catalytic domain located in the lumen of the secretory pathway ( Figure 24A ). Their functional importance for...

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Abstract

Described herein are compositions and methods of producing glycosylated proteins in vitro and in vivo. The methods include using host cells to produce glycosylated proteins. Also described herein areglycosylated proteins produced using such methods and uses thereof.

Description

[0001] 1. Cross-references to related applications [0002] The present application claims priority to U.S. Provisional Patent Application No. 62 / 527,466, filed June 30, 2017, which is incorporated herein by reference in its entirety. [0003] 2. Sequence Listing [0004] The present application contains a Sequence Listing filed in ASCII format and incorporated herein by reference in its entirety. Said ASCII copy, created on June 28, 2018, is named 14197-005-228_ST25_FINAL.txt and is 1,065,529 bytes in size. [0005] 3. Technical field [0006] Compositions and methods for producing glycosylated proteins in vitro and in vivo are described herein. The methods include using host cells to produce glycosylated proteins. Glycosylated proteins produced using these methods and uses thereof are also described herein. [0007] 4. Background technology [0008] Protein glycosylation is a ubiquitous post-translational modification in all domains of life. There is enormous complexity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12N1/00
CPCC12P21/005C07K14/505C07K16/2887C12R2001/90C07K2317/14C07K2317/41C07K14/44C12N9/1051C12N9/1081C07K2319/04C07K2319/05C12N15/79C07K16/00C07K2319/30C12N1/105C12N9/1029C12Y203/01003C12Y204/01022C12Y204/01101C12Y204/01143C12Y204/99006
Inventor 阿米尔礼萨·法里德莫亚耶曼纽拉·玛莉瑞纳·福拉多尔
Owner LIMMATECH BIOLOGICS AG