Processing of an antigenic sequence from IgG constant domains for presentation by MHC class II

Area of Science:

• Immunology
• Protein Engineering
• Antibody Engineering

Background:

• Targeting T cell epitopes to antigen-presenting cells (APCs) is a strategy to enhance T cell responses.
• Antibodies can be engineered to deliver specific payloads, but optimizing their structure for secretion and antigen presentation is crucial.

Purpose of the Study:

• To investigate the impact of substituting loops in human IgG constant H (C(H)) domains with T cell peptide epitopes on antibody secretion and T cell activation.
• To identify optimal locations within IgG domains for epitope insertion to maximize T cell responses.

Main Methods:

• Engineered APC-specific antibodies (anti-IgD) by substituting 18 loops in C(H)1, C(H)2, and C(H)3 domains with a T cell peptide epitope.
• Assessed secretion levels of engineered antibody-epitope fusion molecules.
• Evaluated T cell activation in vitro and in vivo (BALB/c mice) using these engineered antibodies.

Main Results:

• Antibody secretion levels followed a hierarchy: C(H)2 >= C(H)1 > C(H)3, with loop 6 (FG) showing best secretion within each domain.
• Mutants with epitopes in C(H)2 were most effective for T cell activation in vitro, particularly loop 4C(H)2.
• In vivo studies showed loop 4C(H)2 mutants induced specific T cell activation at 100-fold lower concentrations than loop 6C(H)1 mutants.

Conclusions:

• Eight specific loops (all in C(H)2, plus loop 6 in C(H)1 and C(H)3) are suitable for delivering T cell epitopes to APCs.
• The C(H)2 domain, specifically loop 4, is highly effective for enhancing T cell activation both in vitro and in vivo.
• Engineered antibodies offer a promising platform for targeted immune stimulation.