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Fc containing polypeptides with altered glycosylation and reduced effector function

A technology of effectors and glycosylation, applied in chemical instruments and methods, medical preparations of non-active ingredients, peptides, etc., can solve problems such as poor biophysical properties of antibodies

Active Publication Date: 2015-07-22
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, glycosylation of antibody Fc domain is critical for antibody structure, stability and function and glycosylation can lead to poor biophysical properties of antibodies

Method used

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  • Fc containing polypeptides with altered glycosylation and reduced effector function
  • Fc containing polypeptides with altered glycosylation and reduced effector function
  • Fc containing polypeptides with altered glycosylation and reduced effector function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0349] Example 1. Design, preparation and characterization of 2C3 anti-CD-52 highly glycosylated antibody mutants

[0350] Multiple hyperglycosylation mutations are designed in the heavy chain of the anti-CD-52 antibody 2C3 for the purpose of adding large groups to the interaction interface (for example, FcRn binding sites to regulate antibody pharmacokinetics), For adjusting the antibody effector function by changing its interaction with FcγRs, or for introducing new cross-linking site subsequence chemical modification for effector part coupling, the effector part includes but not limited to drugs, toxins , Cytotoxic agents and radionuclides. The highly glycosylated 2C3 mutants are described in Table 3.

[0351] Table 3. Highly glycosylated 2C3 anti-CD-52 mutants

[0352]

[0353] 1A. Generation of 2C3 anti-CD-52 antibody highly glycosylated mutant

[0354] The A114N mutation assigned based on the Kabat numbering system was introduced into the CH1 domain by mutagenic PCR. To gene...

Embodiment 2

[0369] Example 2. Preparation of highly glycosylated mutants in various antibody frameworks

[0370] In addition to the 2C3 anti-CD-52 antibody, the A114N mutation was engineered into multiple antibody backbones to demonstrate that unique hyperglycosylation sites can introduce unrelated heavy chain variable region sequences. The highly glycosylated anti-TEM1, anti-FAP and anti-Her2 mutants are described in Table 5.

[0371] Table 5. A114N and / or S298N mutants designed in a variety of unrelated antibody frameworks

[0372]

[0373] 2A. Generation of highly glycosylated mutants of anti-TEM1 and anti-FAP antibodies

[0374] The A114N mutation assigned based on the Kabat numbering system was introduced into the anti-TEM1 and anti-FAP CH1 domains by mutagenic PCR. To generate a full-length antibody, the mutated VH plus residue 114 was inserted into the pENTR-LIC-IgG1 vector encoding antibody CH domains 1-3 by ligation-independent cloning (LIC). The full-length mutant was then cloned int...

Embodiment 3

[0396] Example 3: Generation of S298N / Y300S Fc mutant

[0397] Design and generate engineered Fc mutants, in which a new glycosylation site is introduced at EU site Ser298, next to the naturally occurring Asn297 site. Glycosylation maintained at Asn297 or eliminated by mutation. The mutations and expected glycosylation results are set out in Table 9.

[0398] Table 9: Glycosylation status of various antibody variants

[0399]

[0400] 3A. Generation of altered glycosylation variants of H66 αβ-TCR antibody

[0401] The mutation was generated on the heavy chain of αβ T-cell receptor antibody clone #66 by using the pENTR_LIC_IgG1 template. The VH domain of HEBE1Δab IgG1#66 was amplified with LIC primers before cloning into mutant or wild-type pENTR_LIC_IgG1 by LIC to generate full-length mutant or wild-type antibodies. The subcloning was verified by DraIII / XhoI double digestion, and an insert fragment with a size of about 1250bp was generated in the successful clone. Those full-leng...

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Abstract

Provided are binding polypeptides (e.g., antibodies), and drug conjugates thereof, comprising an Fc domain with an altered glycosylation profile and reduced effector function. In particular embodiment, the Fc domain comprises: an asparagine residue at amino acid position 298, according to EU numbering; and a serine or threonine residue at amino acid position 300, according to EU numbering. Also provided are nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

Description

[0001] Related application [0002] This application claims to protect the international application number PCT / EP2012 / 003819 filed on September 12, 2012 with the name "anti-αβTCR antibody" and the US provisional application 61 / 776,715 filed on March 11, 2013 with the name " Fc-containing polypeptides with altered glycosylation and reduced effector functions are the priority of the application. [0003] This application is also associated with the U.S. Provisional Application 61 / 776,724 filed on March 11, 2013 under the title "Site-specific antibody drug coupling through glycosyl engineering" and the title filed on March 11, 2013 under the title "High US Provisional Application 61 / 776,710 related to "glycosylated binding polypeptide". The content of the aforementioned application is incorporated in its entirety by reference. Background of the invention [0004] Antibodies with reduced or eliminated Fc glycosylation have been used in the treatment of inflammatory and autoimmune dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28
CPCC07K16/40C07K2317/71C07K2317/92C07K16/2893C07K2317/41C07K16/32C07K16/2809C07K2317/52C07K16/2851A61K47/6803A61P29/00A61P35/00A61P37/00C07K16/28A61P37/02A61P37/06A61K39/00A61K2039/505A61K49/0004C07K2317/73
Inventor C·潘H·邱
Owner GENZYME CORP
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