Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism

Area of Science:

• Immunology
• Oncology
• Biotechnology

Background:

• CXCR4 receptor plays a role in stem and immune cell migration, and is implicated in cancer progression.
• Antagonizing CXCR4 signaling is a therapeutic strategy for various cancers.
• Existing CXCR4 antagonists include small molecules and nanobodies, with potential for improved potency and safety.

Purpose of the Study:

• To compare the affinities of various multivalent nanobody formats targeting CXCR4.
• To investigate the impact of avidity on nanobody performance.
• To develop high-potency CXCR4 antagonistic nanobodies.

Main Methods:

• Generation of multivalent nanobodies with varying valencies (bivalent to decavalent) through fusion to linkers, Fc-regions, or multimerization domains.
• Site-directed conjugation to a trivalent linker scaffold.
• Assessment of nanobody potency in inhibiting CXCL12-mediated Gα_i- or β-arrestin recruitment.

Main Results:

• C-terminal fusion, particularly to human Fc, yielded the most advantageous nanobody formats.
• Fc-fusion resulted in a 2-log-fold and 3-log-fold increase in potency for inhibiting Gα_i- and β-arrestin recruitment, respectively.
• Fc-tail fusion demonstrated the highest avidity effect regardless of the hinge linker.

Conclusions:

• Strategies for generating multivalent, high-potency CXCR4 antagonistic nanobodies were described.
• Nanobodies can induce CXCR4 receptor clustering.
• Fusion to an Fc-tail is an effective strategy for enhancing nanobody avidity and potency against CXCR4.