Cytoplasmic delivery of antibodies through grafting a functional single complementarity-determining region loop

Area of Science:

• Immunology
• Molecular Biology
• Protein Engineering

Background:

• The mouse 3D8 anti-DNA antibody exhibits cell-penetrating properties, localizing in the cytoplasm.
• This cellular uptake is primarily mediated by the complementarity-determining region 1 of the variable light chain (CDR L1).

Purpose of the Study:

• To investigate if grafting the CDR L1 loop from 3D8 onto non-cell-penetrating IgG antibodies can confer cytoplasmic localization ability.
• To determine if antigen-binding activity is retained after CDR L1 grafting.

Main Methods:

• Grafting the CDR L1 loop from the 3D8 antibody onto three different non-cell-penetrating IgG antibodies.
• Assessing cytoplasmic localization and antigen-binding activity of the modified antibodies.
• Utilizing in silico protein modeling to analyze structural contributions to cell penetration.

Main Results:

• One of the three grafted IgGs successfully entered cells and localized in the cytoplasm.
• The modified IgG retained its antigen-binding activity.
• In silico modeling indicated structural similarity between the grafted CDR L1 and the original 3D8 CDR L1 is crucial for cell penetration.

Conclusions:

• Incorporating a specific CDR loop (CDR L1) into an antibody backbone can confer cell-penetrating capability.
• This approach allows for the engineering of antibodies that can enter cells while maintaining their antigen-binding affinity.
• The findings propose a novel strategy for developing cell-penetrating antibodies for various applications.