Optimal design features of camelized human single-domain antibody libraries

Area of Science:

• Biochemistry
• Immunology
• Protein Engineering

Background:

• Antibody variable heavy (V(H)) domains are crucial for antigen binding.
• Camelid V(H) sequences offer unique structural properties for library construction.
• Optimizing V(H) libraries requires understanding antigen contact residues and structural constraints.

Purpose of the Study:

• To construct and validate a human V(H) library using a camelized V(H) scaffold.
• To investigate the role of CDR3 residues in antigen recognition.
• To assess the impact of a CDR1-CDR3 disulfide bridge on V(H) function and stability.

Main Methods:

• Construction of a human V(H) library with randomized CDR3 residues.
• Panning the library against monoclonal antibody targets.
• Mass spectrometry, surface plasmon resonance (SPR), and NMR spectroscopy for structural and binding analyses.
• (1)H-(15)N HSQC spectra to assess protein solubility and aggregation.

Main Results:

• The V(H) library successfully generated V(H) sequences with antigen-specific CDR3 residues.
• C-terminal CDR3 residues were frequently identified as antigen contact sites.
• Disulfide bond formation between CDR1 and CDR3 was observed but not always advantageous for binding.
• The chosen V(H) scaffold facilitated high yields of soluble, non-aggregated proteins.

Conclusions:

• The camelized V(H) scaffold is suitable for constructing functional V(H) libraries.
• Randomization of CDR3 residues is effective for identifying antigen contact sites.
• Disulfide bridging between CDR1 and CDR3 can be explored but requires careful consideration of its impact on antigen binding.