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Anti-cd20 glycoantibodies and uses thereof

a glycoantibody and anti-cd20 technology, applied in the field of anti-cd20 glycoantibodies, can solve the problems of reducing the life span of the circulatory system of the drug, affecting the effector function of the therapeutic antibody, and provoking immunogenicity, so as to improve the therapeutic value, increase the binding affinity of the fc receptor, and increase the activity of the adcc.

Inactive Publication Date: 2015-12-03
ACAD SINIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent is about a new type of antibody called "glycoantibodies" that can target a protein called CD20. These glycoantibodies are made by modifying existing antibodies with a special sugar molecule called "N-glycan". These modifications increase the ability of the antibodies to kill cancer cells and improve their overall therapeutic value. The patent also describes a combination of these glycoantibodies with other drugs or antibodies for use in cancer treatment. These modified antibodies have the potential to be more effective and better tolerated in treating cancer compared to existing drugs.

Problems solved by technology

However, these expression systems suffer from a number of drawbacks that can negatively affect the effector function of therapeutic antibodies.
Their glycosylation machinery often adds undesired carbohydrate determinants which may alter protein folding, induce immunogenicity, and reduce circulatory life span of the drug.
Notably, sialic acid as N-acetylneuraminic acid is not efficiently added in most mammalian cells and the 6-linkage is missing in these cells.
Engineering cells with the various enzymatic activities required for sialic acid transfer has not yet succeeded in providing a human-like pattern of glycoforms to protein drugs.

Method used

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  • Anti-cd20 glycoantibodies and uses thereof
  • Anti-cd20 glycoantibodies and uses thereof
  • Anti-cd20 glycoantibodies and uses thereof

Examples

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example 1

General Procedure for Analysis of N-Glycosylation of Anti-CD20 Antibodies

[0233]We developed a mass spectrometric method to monitor the yield of oligosaccharide-derived fragment ions (oxonium ions) over a collision induced dissociation (CID) energy applied to a glycopeptides precursor. Multiple Reaction Monitoring (MRM) of oxonium ions method could fulfill the regulatory requirement on the routine quality control analysis of forthcoming biosimilar therapeutics.

[0234]5 ug of Rituximab (purchased from Genentech) was dissolved in 25 ul of 2M Guanidine-HCl, and dithiothreitol (DTT) were added to a final concentration of 5 mM. After 10 minutes incubation in 110° C., reduced cysteine residues were alkylated in 10 mM Iodoacetamide (IAA) at 37° C. for 1 hour. Add 5 mM DTT to quench excess IAA at RT for 10 minutes. The product was diluted 15 times in 50 mM ammonium bicarbonate before microcentrifugation with spin column (10 kDa protein MW cut-off). The trypsin digestion was performed for 4 ho...

example 2

Generation of Anti-CD20 GAbs

Anti-CD20 GAb301

[0238]The complete removal of N-linked glycan at Asn297 from Fe region of Rituximab (Rituxan) is achieved by means of PNGase F, and evaluated with 4-12% Bis-Tris NeuPAGE and LC-MS / MS analysis of tryptic glycopeptides from modified and unmodified IgG. The molecular weights of tryptic glycopeptides helps to determine the potential site of N-linked glycosylation at each asparagine and to elucidate the species of predominant glycans.

Anti-CD20 GAb200

[0239]Commercial or home-made heterogeneous mAb was used as the starting material and modified with selected glycosidases. Application of Endoglycosidase (Endo F2, Endo F3, or Endo H) can yield a homogenous di-sugar mAb of GlcNAc-Fuc at its Fc N-linked glycosylation site (GAb200). Subsequently a homogeneous mono-sugar mAb can be obtained with application of fucosidase; or the mono-sugar species can also be obtained with combination of Endo F3 and fucosidase in one step as shown with Rituximab. The p...

example 3

Characterization of GAb301

[0264]Anti-CD20 GAb301 was tested for its antigenic binding and induced functions using B-lymphoma Ramos cells. The sugar-free Rituximab variant retained a full strength in both CD20 binding activity and induction of apoptosis, and reserved a 35% of CDC effect as compared to the Rituximab's maximum values; however, GAb301 lost almost completely the ADCC effect. These results indicate the presence of carbohydrates is essential to the induction of ADCC; the CDC activity is much impaired.

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Abstract

The present disclosure relates to a novel class of anti-CD20 monoclonal antibodies comprising a homogeneous population of anti-CD20 IgG molecules having the same N-glycan on each of Fc. The antibodies of the invention can be produced from anti-CD20 monoclonal antibodies by Fc glycoengineering. Importantly, the antibodies of the invention have improved therapeutic values with increased ADCC activity and increased Fc receptor binding affinity compared to the corresponding monoclonal antibodies that have not been glycoengineered.

Description

RELATED APPLICATION[0001]This application claims the benefit of U.S. provisional applications U.S. Ser. No. 62 / 003,136, May 27, 2014, U.S. Ser. No. 62 / 020,199, Jul. 2, 2014, and U.S. Ser. No. 62 / 110,338, filed Jan. 30, 2015. The contents of each of which is hereby incorporated by reference in its entirety.FIELD[0002]Antibodies, antibody variants, antigen binding fragments and conjugates thereof that bind to CD20 are disclosed herein, as well as related compositions and methods of use. Methods of use include, without limitation, cancer therapies and diagnostics.BACKGROUND OF THE INVENTION[0003]Immunoglobulins and Fc receptors are critical glycoprotein components of the immune system. Fc receptors bind the Fc (effector) region of antibody molecules and communicate information within the innate and adaptive immune systems. Glycosylation of antibodies, particularly in the Fc region of IgG, plays an important role in the modulation of the activity of the antibody. The N-glycans in the id...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12P21/00
CPCC07K16/2887C07K16/283C12P21/005C07K2317/41C07K2317/24C07K2317/31C07K2317/732A61K2039/505A61K2039/54A61K2039/545C07K2317/734C07K2317/92C12Y302/01051C12Y302/01A61P35/00A61P35/02A61P43/00C12N9/2402C07K2317/73
Inventor WONG, CHI-HUEYWU, CHUNG-YITSAI, MING-HUNG
Owner ACAD SINIC
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