Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• B-cell lines with constitutive immunoglobulin V gene hypermutation can be cultured in vitro.
• Generating antigen-specific antibodies with high affinity is crucial for therapeutic and diagnostic applications.

Purpose of the Study:

• To demonstrate the utility of iterative antigen-mediated selection of hypermutating B-cell lines for in vitro antibody generation.
• To explore the role of complementarity-determining regions (CDRs) and framework mutations in antibody affinity maturation.

Main Methods:

• Utilized Ramos (human B-cell line) and a hypermutating chicken B-lymphoma line for antibody selection.
• Employed iterative antigen-mediated selection to drive stepwise improvement in antibody binding affinity.
• Analyzed mutations in CDRs and framework regions contributing to antibody maturation.

Main Results:

• Derived B-cell descendants with stepwise improved binding to streptavidin, initially via CDR mutations and later framework mutations.
• Successfully generated mutated antibodies with increasing affinity to independent test antigens from a single chicken B-lymphoma cell.
• Achieved nanomolar affinities for selected antibodies, starting from an exceedingly low initial affinity threshold.

Conclusions:

• Iterative antigen-mediated selection of hypermutating B-cell lines is an effective strategy for in vitro generation of antigen-specific monoclonal antibodies.
• The method is extendable to the maturation of other protein-ligand interactions.
• Framework mutations play a strategic role in antibody affinity maturation alongside CDR mutations.