Heptavalent scaffold of C4BP
Protein avimers (from avidity multimer) are a class of multidomain proteins possessing binding and inhibitory properties. Multimerization of a signal molecule often leads to the increase of the intensity of the resulted signal. Thus, protein oligomerization has been found to cause higher binding strength, structure stabilization, and allows to combine more than one function in the same molecule. Although synthetic scaffolds ordering and presenting molecular building blocks in a certain way have been well established production of multimeric constructs has been still connected with the necessity to overcome the functional diversity of biologic molecules by the introduction of reactive coupling partners aimed on successive orthogonal ligations.
Combinatorial optimization and avidity modulation upon multimerization
Multivalent interactions of biological molecules play an important role in living systems. A multivalent ligand consists of multiple copies of ligand molecules that are conjugated to scaffolds, allowing for their simultaneous binding to multiple target molecules that for example reside on the surface of cells. Many research groups have successfully generated multivalent ligands to increase the net binding affinity and specificity of the ligand to the receptor [21,22]. Direct peptide oligomerisation or polymeric display of peptides on an oligomeric scaffold is a method that was successfully used to enhance the interaction strength of binding molecules for their target ligands through an avidity effect [23-26].
Oligomerisation via conjugation with neutravidin, fusion to antibody Fc domain, or the oligomerisation domain of C4 binding protein resulted in oligovalent variants that displayed an up to 400-fold improved apparent dissociation constant in the low nanomolar range.
In this study, we demonstrate that MCoTI-II, a trypsin inhibitor from squash can be engineered to bind to CTLA-4, an inhibitory receptor expressed by T lymphocytes that has emerged as a target for the treatment of metastatic melanoma. We compared the avidity effects of various dimeric, tetrameric, and heptameric variants on CTLA-4 binding. Our data indicate that knottin oligomerisation is a valid strategy to obtain miniproteins with significantly improved binding characteristics that may become valuable tools in diagnostics and therapy.
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