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Related Concept Videos

Antibody Structure01:10

Antibody Structure

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Overview
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
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Antibody Structure and Classes01:25

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Antibodies, also known as immunoglobulins, are produced by B cells in response to foreign substances, such as bacteria and viruses. These proteins are critical for recognizing and neutralizing these substances, protecting the body from potential harm.
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Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
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Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
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Engineering a human IgG2 antibody stable at low pH.

Seiji Saito1, Hiroshi Namisaki2, Keiko Hiraishi1

  • 1Antibody & Biologics Research Laboratories, R&D Division, Kyowa Kirin Co., Ltd., Tokyo, Japan.

Protein Science : a Publication of the Protein Society
|March 7, 2020
PubMed
Summary
This summary is machine-generated.

Engineered IgG2 antibodies with an IgG1 CH2 domain show reduced aggregation during low pH processing. Specific amino acid substitutions further enhance stability, improving therapeutic antibody design.

Keywords:
ADCCCDCIgG2 subclassacid-induced aggregateaggregationantibodysubclass change

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Area of Science:

  • Biochemistry
  • Immunology
  • Biotechnology

Background:

  • Immunoglobulin G2 (IgG2) antibodies possess unique characteristics, including limited effector functions and a rigid hinge region.
  • Despite clinical applications, IgG2 antibodies are prone to aggregation, especially under acidic conditions during bioprocessing, which can compromise their efficacy and increase immunogenicity.

Purpose of the Study:

  • To engineer a more stable IgG2 antibody by modifying its constant domains.
  • To investigate the impact of replacing the IgG2 constant domain with an IgG1 constant domain on antibody aggregation.
  • To identify specific amino acid substitutions within the CH2 domain that enhance IgG2 stability.

Main Methods:

  • Replaced the constant domain of an anti-2,4-dinitrophenol (DNP) IgG2 antibody with the IgG1 constant domain.
  • Introduced specific amino acid substitutions (F300Y, V309L, T339A) into the CH2 domain, creating the IgG2_YLA variant.
  • Assessed antibody aggregation at low pH using various analytical techniques.
  • Determined the thermal stability of the CH2 domain using differential scanning calorimetry.
  • Evaluated antigen-binding capacity, FcγRIIIa affinity, and C1q binding ability.

Main Results:

  • The IgG2 antibody with the IgG1 CH2 domain exhibited reduced aggregation at low pH compared to the original IgG2.
  • The IgG2_YLA variant showed significantly decreased aggregation at low pH and an increased CH2 transition temperature.
  • IgG2_YLA maintained similar antigen-binding capacity to IgG2 while retaining low affinity for FcγRIIIa and C1q.
  • The YLA substitution also reduced low pH-induced aggregation in panitumumab, another IgG2 antibody.

Conclusions:

  • Engineering the IgG2 constant domain, particularly the CH2 region, can significantly improve antibody stability during bioprocessing.
  • Specific amino acid substitutions (YLA) offer a promising strategy for developing more robust IgG2-based therapeutics.
  • These findings provide a foundation for designing next-generation IgG2 antibodies with enhanced stability and therapeutic potential.