Llamanade : an open-source computational pipeline for robust nanobody humanization.

Area of Science:

• Biotechnology
• Immunology
• Computational Biology

Background:

• Nanobodies (Nbs) are promising antibody fragments for therapeutics but require humanization due to their camelid origin.
• Existing methods lack systematic approaches for rational Nb humanization.
• There is a need for dedicated software to guide Nb humanization for improved clinical translation.

Approach:

• Systematic analysis of Nb sequence and structural properties using next-generation sequencing (NGS) databases and high-resolution structures.
• Development of Llamanade, a user-friendly, open-source computational pipeline for Nb humanization.
• Validation of Llamanade using SARS-CoV-2 neutralizing nanobodies, assessing humanness, solubility, and bioactivity.

Key Points:

• Identified conserved residues contributing to Nb solubility, stability, and antigen-binding, informing humanization strategies.
• Llamanade provides sequence features, model structures, and optimized humanization solutions.
• Humanized Nbs using Llamanade showed increased humanness, resembling human IgGs, with preserved solubility and bioactivity.

Conclusions:

• Llamanade facilitates rational nanobody humanization, enhancing translational potential for therapeutic development.
• The tool supports the creation of safer and more effective nanobody-based therapeutics.
• Llamanade is freely available and will be accessible via a web server to aid the expanding field of therapeutic nanobodies.