Nanobody Humanization (Llamanade)

1 Introduction

Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite their notable physicochemical properties, Nbs, which are derived from camelids, may require “humanization” to enhance their translational potential for clinical trials. The authors of Llamanade systematically analyzed the sequence and structural properties of Nbs based on next-generation sequencing (NGS) databases and high-resolution structures. Their analysis revealed substantial framework diversities and highlighted key differences between Nbs and human Immunoglobulin G (IgG) antibodies. They identified conserved residues that may contribute to improved solubility, structural stability, and antigen-binding, providing valuable insights into the humanization of Nbs.

Based on their big data analysis, the authors developed "Llamanade"¹, a user-friendly, open-source tool designed to facilitate the rational humanization of Nbs. By using an Nb sequence as input, Llamanade provides information on sequence features, model structures, and optimized solutions for humanizing Nbs. The full analysis for a given Nb can be completed in less than a minute on a local computer. To demonstrate the robustness of this tool, the authors successfully applied it to humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server, supporting the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective clinical trials.

Figure 1. Schematic pipeline of Llamanade.

2 Performance

Figure 2. The humanization of SARS-Cov-2 Nbs by Llamanade and verification.

To demonstrate the robustness of Llamanade on nanobody humanization, authors humanized nine highly potent SARS-CoV-2 neutralizing Nbs that have been identified². These Nbs target the RBD of the virus spike and are structurally diverse, falling into three main epitope classes³. Using sequence as input, these RBD Nbs were humanized by Llamanade, which significantly improved the humanness level of Nb frameworks. The median T20 score⁴ was substantially increased from 82.8 (before humanization) to 92.4 (after humanization). To assess the bioactivities of these humanized Nbs, ELISA was performed and confirmed the comparably high activities (±4-fold) to the non-humanized precursors (Figures 2D).

3 Parameters

4 Results Explanation

result：

The sequence and humanness score(T20 score) of both the original and humannized nanobody will be provided in the format below:

pdb file:

In addition, we also provide the 3D structure PDB file of the original nanobody, , which can be obtained in two ways:

1. If the user does not provide a PDB file, the Llamanade tool will use the modeling method to predict the structure of the original nanobody and output it as a PDB file;
   
2. If the user provides a PDB file and specifies the chain of the nanobody within it, the Llamanade tool will extract the nanobody from the input PDB and output it as a new PDB file.

5 Reference

[1] Sang Z, Xiang Y, Bahar I, Shi Y. Llamanade: An open-source computational pipeline for robust nanobody humanization. Structure. 2022 Mar 3;30(3):418-429.e3. doi: 10.1016/j.str.2021.11.006. Epub 2021 Dec 10. PMID: 34895471. https://doi.org/10.1016/j.str.2021.11.006
[2] Xiang, Y., Nambulli, S., Xiao, Z., Liu, H., Sang, Z., Duprex, W.P., Schneidman Duhovny, D., Zhang, C., and Shi, Y. (2020). Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2. Science 370, 1479–1484. https://doi.org/10.1126/science.abe4747
[3] Sun, D., Sang, Z., Kim, Y.J., Xiang, Y., Cohen, T., Belford, A.K., Huet, A., Conway, J.F., Sun, J., Taylor, D.J., et al. (2021). Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting diverse and conserved epitopes. Nat. Commun. 12, 4676. https://doi.org/10.1038/s41467-021-24963-3
[4] Gao, S.H., Huang, K., Tu, H. et al. Monoclonal antibody humanness score and its applications. BMC Biotechnol 13, 55 (2013). https://doi.org/10.1186/1472-6750-13-55