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Site-specific antibody-drug conjugation through glycoengineering

A technology of glycans and linkers, applied in the field of site-specific antibody-drug conjugation through glycoengineering, can solve problems such as coupling difficulties and antibody affinity reduction.

Active Publication Date: 2016-01-06
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, many lysines are located in the important antigen binding sites of CDR regions and drug conjugation may lead to the decrease of antibody affinity
Elsewhere, although sulfhydryl-mediated coupling primarily targets the eight cysteines involved in hinge disulfide bonds, prediction and identification of which four of the eight cysteines are consistently coupled across preparations still very difficult

Method used

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  • Site-specific antibody-drug conjugation through glycoengineering
  • Site-specific antibody-drug conjugation through glycoengineering
  • Site-specific antibody-drug conjugation through glycoengineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 12C3

[0365] Example 1. Design, preparation and characterization of 2C3 anti-CD-52 highly glycosylated antibody mutants

[0366] For the purpose of adding large groups to the interaction interface (e.g. FcRn binding site to modulate antibody pharmacokinetics), for modulating antibody effector functions by altering its interaction with FcγRs, or for introducing novel cross-linking sites Point subsequence chemical modification for conjugation of effector moieties including but not limited to drugs, toxins, cytotoxic agents and radionuclides, multiple hyperglycosylation mutations were designed in the heavy chain of anti-CD-52 antibody 2C3. The highly glycosylated 2C3 mutants are illustrated in Table 3.

[0367] Table 3. Highly glycosylated 2C3 anti-CD-52 mutants

[0368]

[0369] 1A. Generation of hyperglycosylated mutants of 2C3 anti-CD-52 antibody

[0370] The A114N mutation, referred based on the Kabat numbering system, was introduced into the CH1 domain of 2C3 by mutagenesis...

Embodiment 2

[0384] Example 2. Preparation of highly glycosylated mutants in several antibody backbones

[0385] In addition to the 2C3 anti-CD-52 antibody, the A114N mutation was engineered in several other antibody backbones to identify unique hyperglycosylation sites that could be introduced into unrelated heavy chain variable domain sequences. The highly glycosylated anti-TEM1, anti-FAP and anti-Her2 mutants are set forth in Table 5.

[0386] Table 5. A114N and / or S298N mutants designed in several unrelated antibody frameworks

[0387]

[0388] 2A. Generation of anti-TEM1 and anti-FAP antibody hyperglycosylation mutants

[0389] The A114N mutation designed based on the Kabat numbering system was introduced into the anti-TEM1 and anti-FAP CH1 domains by mutagenesis PCR. To generate the full-length antibody, the mutated VH plus residue 114 was inserted by ligation-independent cloning (LIC) into the pENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. The full-length mutants we...

Embodiment 3

[0409] Example 3: Generation of S298N / Y300SFc mutants

[0410] Engineered Fc variants were designed and generated in which a new glycosylation site was introduced at EU position Ser298, adjacent to the naturally occurring Asn297 site. Glycosylation at Asn297 was maintained or eliminated by mutation. Mutations and expected glycosylation results are set forth in Table 9.

[0411] Table 9: Glycosylation status of various antibody variants

[0412]

[0413] 3A. Generation of altered glycosylation variants of the H66αβ-TCR antibody

[0414]Mutations were performed on the heavy chain of αβ T-cell receptor antibody clone #66 by Quikchange using the pENTR_LIC_IgG1 template. The VH domain of HEBE1ΔabIgG1#66 was amplified with LIC primers, followed by LIC cloning into mutant or wild-type pENTR_LIC_IgG1 to generate full-length mutant or wild-type antibodies. Subcloning was confirmed with DraIII / XhoI double digestion, yielding an approximately 1250 bp-sized insert in successful c...

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Abstract

The current disclosure provides binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof (e.g., antibody-drug conjugates or ADCs), comprising a site- specifically engineered drug-glycan linkage within native or engineered glycans of the binding polypetpide. The current disclosure also provides 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 U.S. Provisional Application 61 / 776,724, filed March 11, 2013, entitled "Site-Specific Antibody Drug Conjugation by Glycoengineering," and filed March 11, 2013, entitled "Highly Glycosyl U.S. Provisional Application 61 / 776,710, entitled "Fc-Containing Polypeptides Having Altered Glycosylation and Reduced Effector Function," filed March 11, 2013. The content of the aforementioned application is hereby incorporated by reference in its entirety. Background of the invention [0003] The treatment of cancer is still a great challenge for human beings. Although current standard therapies including surgery, radiotherapy, and chemotherapy have saved the lives of many patients, there is still a great need for more effective therapies, especially target-specific therapies with higher efficacy and better therapeutic windows. These target-specific therapies employ antibody-drug conjugates (ADCs), in which antigen-specific antib...

Claims

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Application Information

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IPC IPC(8): A61K39/00C07K16/00
CPCC07K2317/40C07K2317/41C07K2317/522C07K2319/00C07K16/2809C07K16/2851C07K16/32C07K16/40C07K2317/56C07K2317/732C07K2317/92C07K2317/71C07K2317/94A61K47/6889A61K47/6869A61K47/549C07K16/2893A61P35/00A61P43/00A61K47/68031C07K2317/76C07K16/28A61K2039/505A61K47/6803
Inventor C·潘Q·周J·斯特凡诺P·达尔B·陈D·贾诺利奥R·米勒H·邱
Owner GENZYME CORP
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