Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG₁ antibodies

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Bispecific antibodies, particularly bispecific IgG, represent a significant advancement in therapeutic options.
• Engineering strategies are crucial for the efficient production of bispecific IgG for clinical applications.

Purpose of the Study:

• To assess the immunogenicity risk associated with engineering strategies used for bispecific IgG production.
• To evaluate the utility of research-grade IgG1 antibodies as surrogates for immunogenicity risk assessment.

Main Methods:

• Construction and assessment of monospecific and bispecific IgG1 antibodies.
• Utilizing assays such as dendritic cell internalization, T cell proliferation, and T cell epitope identification.
• Employing in silico prediction and MHC-associated peptide proteomics for epitope analysis.

Main Results:

• Knobs-into-holes (KIH) mutations and in vitro assembly of half antibodies do not pose a major immunogenicity risk.
• Fab mutations for in vivo assembly of bispecifics in single host cells also show no significant increase in risk.
• Research-grade preparations of trastuzumab and bevacizumab exhibited comparable or slightly higher immunogenicity risk compared to Herceptin and Avastin, respectively.

Conclusions:

• Engineering strategies like KIH and Fab mutations are generally safe regarding immunogenicity for bispecific IgG1.
• Research-grade IgG1 antibodies can be effectively used as surrogates for the immunogenicity risk assessment of their pharmaceutical counterparts.