Abstract
The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Single-domain VHH antibodies exhibit high specificity and affinity but have higher stability and solubility than conventional antibodies as they are single-domain proteins. In this work, based on physicochemical measurements and molecular dynamics (MD) simulations, we have gained insight that will facilitate rational design of single-chain VHH antibodies. We first assessed two homologous VHH antibodies by differential scanning calorimetry (DSC); one had a high (64.8 °C) and the other a low (58.6 °C) melting temperature. We then generated a series of the variants of the low stability antibody and analyzed their thermal stabilities by DSC and characterized their structures through MD simulations. We found that a single mutation that resulted in 8.2 °C improvement in melting temperature resulted in binding affinity an order of magnitude lower than the parent antibody, likely due to a shift of conformational space explored by the single-chain VHH antibody. These results suggest that the delicate balance among conformational stability, binding capability, and conformational space explored by antibodies must be considered in design of fully functional single-chain VHH antibodies.
Highlights
The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors
Our results showed that there is a delicate balance among thermal stability, binding affinity, and explored conformational space that must be considered in engineering of single-domain VHH antibodies
This study focused on two singledomain VHH antibodies, Z18 and Z26, that were selected in-house for affinity to human serum albumin (HSA)
Summary
The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Previous studies demonstrated that single-domain antibodies can be engineered to improve the thermal and colloidal stability[4,34,35] In one such example, based on molecular dynamics (MD) simulations, Bekker et al showed that the fraction of native contacts, or Q-value, that had been employed in studies of protein folding[36] could be used as an evaluation metric to identify residues important for thermal stability of single-domain a ntibodies[37]. Zabetakis et al demonstrated that the stability-enhancing mutations identified by Bekker et al led to reductions of the binding affinities to a ntigen[38] This demonstrated the difficulty of simultaneously improving thermal stability and binding affinity and showed that our understanding of the relationships between binding capability and other physical properties is not yet sufficient to design antibodies in a rational manner
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