Antibody Fragments Humanization: Beginning with the End in Mind

Area of Science:

• Biotechnology
• Immunology
• Molecular Engineering

Background:

• Monoclonal antibodies can be engineered into smaller antigen-binding fragments (e.g., scFvs, Fabs) for in vivo applications, avoiding Fc-mediated functions.
• Rodent-derived antibody fragments often elicit immunogenicity in humans, necessitating humanization for clinical approval.
• Current strategies lack a defined method for producing stable, soluble, humanized antibody V-domains that retain parental antigen-binding properties.

Purpose of the Study:

• To develop a robust strategy for creating humanized antibody V-domains with reduced immunogenicity.
• To maintain high stability and solubility of engineered antibody fragments.
• To preserve the original antigen-binding characteristics of parental antibodies.

Main Methods:

• Grafting complementary determining regions (CDRs) from parental antibodies onto selected human antibody frameworks.
• Utilizing a flowchart for a systematic approach, including introducing affinity purification tags.
• Employing structural refinements to preserve antigen-binding capabilities.
• Implementing in silico analysis, research-scale production, functional evaluation, purification, aggregation, and stability assessments for candidate selection.

Main Results:

• A defined strategy for generating humanized antibody V-domains was established.
• The proposed method facilitates the reduction of immunogenicity.
• The approach allows for the preservation of antigen-binding characteristics, stability, and solubility.

Conclusions:

• The developed CDR-grafting strategy offers a viable method for producing humanized antibody V-domains.
• This approach addresses the limitations of existing methods for creating immunogenic-free antibody fragments.
• The strategy supports the development of novel antibody-based therapeutics with improved safety and efficacy profiles.