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Glycosylation engineered antibody therapy

A glycosylation, antibody technology, applied in fucosylation and ,N-, can solve the problems of unstable expression efficiency and low host system

Inactive Publication Date: 2009-06-24
UNIV OF MARYLAND BALTIMORE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, pre-existing methods of intracellular glycoengineering lead to the generation of heterogeneous mAbs with various glycosylation states
In addition, dramatic genetic engineering of expression systems can result in host system instability and inefficient expression

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Detection of FcgRIIIa receptor (CD16a) and FcgRIIa (CD32) allelic polymorphism

[0086] To determine the ability of glycoengineered mAbs to induce ADCC in patients with various genotypes or to determine the responsiveness of non-glycoengineered mAbs, the allelic variants at position 131 of FcgRIIa and position 158 of FcgRIIIa were characterized using, for example, a PCR-based strategy. body. First, genomic DNA isolated from human tumor cell lines, human saliva, human PBMC, or paraffin-embedded tissue was used as a template for PCR amplification.

[0087] A. Detection of polymorphisms in the FcgIIIa receptor (CD16a) allele using PCR amplification and restriction enzyme digestion.

[0088] Primer design was performed according to the sequence available in GenBank (accession number X52645 for FcgRIIIa, Nieto et al., 2000). This protocol uses a primer that introduces a new RsaI site to one end of all amplification products and a second primer that creates a new...

Embodiment 2

[0094] Prophetic Example 2: Homogeneous Preparation of Antibodies.

[0095] To obtain homogenous preparations of mAbs with specific glycosylation states, systems combining high cell expression yields and in vitro glycoengineering using chemoenzymatic transglycosylation were utilized [27-30]. Combined with the power of chemically synthesizing oligosaccharide oxazoline substrates for endonucleases, this method allows the preparation of a range of defined glycosylation states (native or non-native) of mAbs or their IgG-Fc domains, which in turn allows the systematic Analysis of the structure-activity relationship of IgG glycosylation and ADCC activity. Following the seminal work of Jeffries et al., using hingeless human IgG-Fc i.e. (aa231-447) as a model system in which the hinge region of the Fc has been deleted [7, 31]. Using this truncated Fc form rather than fully human antibody IgG or IgG-Fc as a model system greatly simplifies synthesis and subsequent study of structure-...

Embodiment 3

[0097] Embodiment 3: Design and synthetic example of sugar oxazoline.

[0098]ENGases are a class of endoglycosidases that hydrolyze β-1,4-glycosidic linkages in the core N,N'-diacetylchitobiose moiety of N-glycoproteins to release the N-glycans. However, some ENG enzymes, such as endo-A of Arthrobacter protophormiae and endo-M of Mucor hiemalis, possess transglycosylation activity and are able to convert released N-glycans Transfer to GlcNAc-peptide receptors to form new glycopeptides. Endo-A and endo-M can transfer large intact oligosaccharides to GlcNAc-peptide acceptors in one step to form new glycopeptides, allowing highly focused synthesis of glycopeptides without protecting groups. Chemoenzymatic methods suffer from low transglycosylation yields (typically 5-20%), product hydrolysis, and the limitations of using only native N-glycans as donor substrates. To address these issues, we used synthetic oligosaccharide oxazolines (mimetics of putative oxazolinium ion interme...

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PUM

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Abstract

The instant invention is drawn to methods of generating a glycosylation- engineered antibody, and using the glycosylation-engineered antibody for treating a patient, particularly a cancer patient or a patient with an immune disease or disorder. The instant invention is also drawn to methods of generating a glycosylation-engineered antibody for use in the treatment of patients having a polymorphism that does not respond to conventional antibody therapy. The instant invention is also drawn to methods of improving the biological activity of an antibody by glycosylation engineering. The instant invention is also drawn to methods of modulating antibody-dependent cell-mediated cytoxicity (ADCC) using a glycosylation- engineered antibody.

Description

[0001] Cross References to Related Applications [0001] This application claims the benefit of U.S. Provisional Application 60 / 812,322, entitled "Fc Receptor Polymorphisms in Solid Tumors as Prognosis with Antibody-Mediated Therapy," filed June 9, 2006, and claims January 2007 Benefit of U.S. Provisional Application 60 / 897,966, entitled "Glycoengineered Antibody Therapeutics," filed on 29. The contents of these priority documents are hereby incorporated by reference in their entirety. Background technique [0002] Monoclonal antibodies (mAbs) have emerged as an important class of therapeutic agents for the treatment of human diseases such as cancer [1, 2]. The mAbs currently used in cancer therapy are of the IgG type and are produced in mammalian cells (CHO cells or mouse NSO cell lines, etc.). Following antigen recognition and binding to targets such as tumor cells, mAbs can trigger various effector functions including: 1) antibody-dependent cell-mediated cytotoxicity (ADC...

Claims

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Application Information

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IPC IPC(8): A61K39/00
Inventor S·斯特罗姆L·-X·王
Owner UNIV OF MARYLAND BALTIMORE
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