High resolution functional analysis of antibody-antigen interactions

Area of Science:

• Immunology
• Protein Science
• Biochemistry

Background:

• Monoclonal antibodies (MAbs) targeting human growth hormone (hGH) are crucial for research and therapeutic applications.
• Understanding the precise binding interactions between MAbs and hGH is essential for characterizing their specificity and efficacy.

Purpose of the Study:

• To comprehensively map the functional epitopes of 21 anti-hGH mouse monoclonal antibodies (MAbs) on human growth hormone (hGH).
• To identify the key amino acid side-chains on hGH that are critical for MAb binding and to understand the nature of these antigenic sites.

Main Methods:

• Utilized a combination of homolog-scanning and alanine-scanning mutagenesis to systematically alter hGH side-chains.
• Employed robot-aided enzyme-linked immunosorbent assays (ELISAs) to quantify the binding of MAbs to mutated hGH variants.
• Integrated mutagenesis data with structural information to define high-resolution functional epitopes.

Main Results:

• Each functional epitope comprised at least two spatially proximate polypeptide segments on the folded hGH structure, forming distinct binding patches.
• Identified specific amino acid side-chains, particularly Arg, Pro, Glu, Asp, Phe, and Ile, as dominant contributors to MAb binding.
• Demonstrated that while functional epitopes could overlap, each MAb exhibited a unique binding profile, indicating differential recognition of hGH.

Conclusions:

• The accessible surface of hGH is largely antigenic in mice, with functional epitopes being dominated by a limited number of critical side-chains.
• These findings provide a high-resolution map of hGH-MAb interactions, crucial for developing targeted immunotherapies and diagnostic tools.