Evading pre-existing anti-hinge antibody binding by hinge engineering.

Area of Science:

• Immunology
• Protein Engineering
• Biochemistry

Background:

• Antibody fragments (Fab and F(ab )2) offer therapeutic advantages but can elicit unwanted anti-hinge antibody (AHA) responses.
• Proteolytic cleavage of antibodies can generate neoepitopes, leading to AHA that may reintroduce Fc-like functions, which is undesirable for therapeutic fragments.

Purpose of the Study:

• To engineer antibody fragments with minimal pre-existing AHA reactivity for therapeutic and diagnostic applications.
• To identify specific hinge region modifications that allow efficient proteolytic cleavage while avoiding unwanted AHA binding.

Main Methods:

• Investigated proteolytic cleavage of modified antibody lower hinge regions using IdeS protease.
• Analyzed the reactivity of pre-existing human serum antibodies against upper hinge region variants of human IgG1, IgG2, and IgG4.

Main Results:

• A lower hinge truncation was identified that yields F(ab )2 fragments with no detectable pre-existing AHA binding.
• Human IgG1 upper hinge variants showed significant pre-existing AHA reactivity, unlike IgG2 and IgG4.
• A T225L variant and D221 residue in the upper hinge demonstrated minimal serum reactivity.

Conclusions:

• Engineered antibody fragments can be produced with minimal pre-existing AHA reactivity.
• Specific modifications in the hinge region, particularly for IgG1, are crucial for developing therapeutic and diagnostic antibody fragments.
• This work provides a practical strategy for producing Fab and F(ab )2 with reduced immunogenicity due to pre-existing AHA.