Abstract
Single domain antibodies (sdAbs), made of natural single variable regions of camelid or cartilaginous fish antibodies, or unpaired variable regions of mouse or human IgGs, are some of the more promising biologic modalities. However, such conventional sdAbs have difficulties of either using unwieldy animals for immunization or having high affinity deficiencies. Herein, we offer a versatile method to generate rabbit variable domain of heavy chain (rVH) derived sdAbs with high affinities (KD values of single digit nM or less) and enhanced thermal stabilities (equal to or even higher than those of camelid derived sdAbs). It was found that a variety of rVH binders, including those with high affinities, were efficiently acquired using an rVH-displaying phage library produced at a low temperature of 16 °C. By a simple method to introduce an additional disulfide bond, their unfolding temperatures were increased by more than 20 °C without severe loss of binding affinity. Differential scanning calorimetry analysis suggested that this highly efficient thermal stabilization was mainly attributed to the entropic contribution and unique thermodynamic character of the rVHs.
Highlights
Introduction of additional disulfide bond torabbit variable domain of heavy chain (rVH).Aiming for thermal stability enhancements of the rVHs, we considered the introduction of an additional disulfide bond between residues 54 and 78 (Gly/Ala and Ile, blue-colored in Fig. 2a and b) based on the previous successful results in VHHs22–24
We used a total of 1.9 × 108 spleen and lymph node cells to amplify VH genes from these rabbits. rVH genes were amplified with designed primers (Supplementary Table S1) and inserted into phagemid vector
As correlation was observed for representative five rVHs between binding efficiencies of Enzyme-Linked ImmunoSorbent Assay (ELISA) (Supplementary Fig. S4) and dissociation constants of surface plasmon resonance (SPR) (Table 1), we consider that other ELISA binders such as H2-2-1 or H3-14, which are as efficient as H2-2-2 and H3-15, could have smaller dissociation constants as those of H2-2-2 and H3-15
Summary
Aiming for thermal stability enhancements of the rVHs, we considered the introduction of an additional disulfide bond between residues 54 and 78 (Gly/Ala and Ile, blue-colored in Fig. 2a and b) based on the previous successful results in VHHs22–24. Because VHHhCG was thermally stabilized by C54-C78 without loss of binding activity[22], the introduction of C54-C78 was unlikely to have a negative impact on the rVHs’ binding affinities to antigens. Based on these considerations, in this study we attempted to introduce an additional disulfide bond into the wild type rVHs by Cys mutation of residues 54 and 78. Peptide identification was conducted with MassLynx Mass Spectrometry Software ver. 4.1 and BiopharmaLynx Software ver. 1.3 (Waters Corporation)
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