Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Bispecific antibodies (BsAbs) are advanced therapeutics.
• Fusion of anticalins to monoclonal antibodies (mAbs) offers a cost-effective method for creating novel BsAbs.

Purpose of the Study:

• To evaluate the impact of anticalin fusion position on BsAb functionality.
• To assess the affinity and functional properties of novel anticalin-fused BsAbs.

Main Methods:

• Anticalins targeting human fibronectin were fused to trastuzumab- (H0) or rituximab- (R0) biosimilar mAbs.
• Eight BsAbs were generated with anticalins in N-terminal and C-terminal positions on heavy and light chains.
• Affinity, glycosylation, shape, yield, antigen recognition, and Fc receptor-mediated functions were evaluated.

Main Results:

• Anticalin fusion did not alter BsAb glycosylation, shape, or yield.
• Antigen recognition (Her2/CD20) and fibronectin binding were reduced in certain anticalin positions.
• Fc receptor-mediated functions (CD16 activation, CD32 phagocytosis, FcRn transcytosis) were less efficient with specific anticalin placements, particularly for R family BsAbs.

Conclusions:

• Anticalin fusion is a viable strategy for rapid BsAb generation.
• The position of anticalin fusion critically affects BsAb functionality and Fc receptor interactions.
• Optimizing anticalin placement is essential for developing effective bispecific antibody therapeutics.