# Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition

## TL;DR

Researchers created a synthetic library to generate humanized nanobodies that inhibit IL-6, a key cytokine in inflammation and cancer, without using animal immunization.

## Contribution

A novel synthetic phage display library was developed to rapidly produce high-affinity, humanized nanobodies without animal immunization.

## Key findings

- The library produced humanized nanobodies with high function and intracellular stability using eGFP as a benchmark.
- A standout nanobody, NbL3, inhibited IL-6-enhanced migration in MCF-7 breast cancer cells at low concentrations.
- NbL3 demonstrated high affinity (22.16 nM) and stability, offering a promising therapeutic alternative for IL-6-targeted interventions.

## Abstract

Interleukin-6 (IL-6) is a cytokine that can bind to IL-6 receptor and induce pleiotropic effects. It serves as a critical biomarker, involved in inflammation amplification, tumor progression, and many other disease developments. Nanobodies, featuring small structure and high affinity, are a powerful and versatile tool in medical diagnostics and therapeutics. Here, based on a scaffold optimized for humanization and stability, we developed a synthetic phage display library that rapidly generated high-affinity and humanized nanobodies, negating the need for animal immunization. Using enhanced green fluorescent protein (eGFP) as a benchmark, we demonstrated that the library produced humanized nanobodies with high function and great intracellular stability. The library was then subjected to screening against IL-6. We identified a standout nanobody, NbL3, which exhibited high affinity (22.16 nM) and stability and significantly inhibited IL-6-enhanced migration on the human breast cancer cell MCF-7 at a relatively low concentration. NbL3’s strong blocking activity provides a promising therapeutic alternative for the IL-6-targeted intervention strategy, underscoring the broader potential of our synthetic library as a versatile platform for the development of humanized nanobodies against multiple antigens.