Allosteric Regulation of Antibody Affinity

Single-chain fragment variable (scFv) antibody fragments are constructed by connecting VH and VL domains with a flexible polypeptide linker. Due to their high target specificity and affinity, they are indispensable in research and diagnostics and serve as building blocks in various therapeutic applications, including chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs). However, despite their remarkable potency, scFv-based immunotherapy approaches are associated with life-threatening side effects such as cytokine release syndrome (CRS) and on-target, off-tumor toxicity. Thus, tunable activity through affinity regulation could enhance safety profiles and expand the therapeutic window. This study describes the successful generation of allosterically switchable scFv fragments by replacing the (GGGGS)3 linker of anti-c-myc epitope tag scFv TUN219-2C1 with N- and C-terminally truncated and circularly permutated calmodulin (CaM) variants based on earlier work by Kellmann et al. (2017). The concept rests on the hypothesis that binding of Ca2+ and peptide ligands leads to conformational changes in the CaM linker, which in turn modulates the VH/VL orientation of the scFv-CaM fusion protein. Here, two distinct mechanisms regulating scFv-CaM antigen binding were identified, with truncated CaM linkers emerging as particularly promising switching modules. First, the binding of M13 Var5 switch peptide to the CaM linker induced a rapid transition to predominantly dimeric scFv-CaM molecules as confirmed by SEC and mass photometry. This dimerization led to a more than 20-fold improvement in KD due to avidity effects and was reversible upon Ca2+ stripping. Protein structure prediction using AlphaFold 3 suggested that the observed diabody transition is driven by a substantial reduction in the distance of residues in the linker fusion region. Here, truncating the CaM linker fusion region proved to be an effective strategy for transmitting sufficient mechanical force to destabilize the VH/VL association. Second, BLI measurements revealed a Ca2+-dependent monovalent modulation of antibody-antigen binding kinetics beyond KD, potentially arising from structural changes in the paratope. In the Ca2+-free state, scFv-CaM antibodies exhibited an up to 17-fold faster dissociation (koff) from the c-myc tag antigen, whereas an increased association rate (kon) partially offset overall affinity changes. This approach could serve as a universal platform technology to rapidly convert virtually any scFv fragment into an affinity-regulatable antibody construct, eliminating the need for paratope engineering and could enable the development of novel regulatable scFv-based immunotherapies. Moreover, the allosteric affinity switch may be applied in affinity chromatography for the mild purification of sensitive biomolecules.