Evolution of nanobodies specific for BCL11A

Area of Science:

• Molecular Biology
• Protein Engineering
• Gene Regulation

Background:

• Transcription factors (TFs) regulate genes crucial for human health, but targeting them intracellularly is difficult due to protein disorder.
• BCL11A is a validated therapeutic target for hemoglobin disorders, necessitating specific intracellular reagents.
• Intracellular antibodies, like nanobodies, can probe protein function and validate therapeutic targets within cells.

Purpose of the Study:

• To optimize nanobodies targeting the BCL11A transcription factor for therapeutic applications.
• To develop a method for targeted protein degradation (TPD) of BCL11A in erythroid cells.
• To assess the specificity of engineered nanobodies against BCL11A and its paralog BCL11B.

Main Methods:

• Utilized yeast surface display to generate first-generation nanobodies against BCL11A's zinc finger region (ZF456).
• Employed error-prone mutagenesis, structural determination, and molecular modeling to enhance nanobody binding affinity.
• Validated nanobody-mediated TPD of BCL11A and measured fetal hemoglobin (HbF) reactivation in erythroid cells.

Main Results:

• Engineered nanobodies specifically recognized BCL11A's ZF6 domain.
• Achieved targeted protein degradation of BCL11A in erythroid cells, leading to increased fetal hemoglobin (HbF) expression.
• Demonstrated that evolved nanobodies could differentiate BCL11A from the highly similar BCL11B paralog.

Conclusions:

• Nanobody-mediated TPD is a viable strategy for targeting transcription factors like BCL11A.
• This approach enables the reactivation of fetal hemoglobin (HbF), offering potential for treating hemoglobin disorders.
• Engineered nanobodies provide exquisite specificity, suitable for dissecting TF-gene regulatory networks and validating therapeutic targets.