Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities

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 (K D 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.

Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function

“…As described above, AvidCARs were successfully engineered using an EGFR-targeting rcSso7d-based binder and an affibody directed against HER2. Of note, similar dimerization-dependent AvidCAR activation was also observed when using low-affinity nanobodies targeting green fluorescent protein (GFP) 49 and HER2 50 , (Supplementary Fig. 12 ).…”

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

“…Since a high diversity of VH CDR3 loops is sufficient to provide antigen specificity of any Ab, and this region is a key determinant of antigen recognition [ 39 ], the functional role of such “artificial” VL fragment in scFv molecules may be associated with an increase in their thermal stability and a decrease in aggregation tendencies. In the case of sdAbs it is ensured by a special repertoire of hydrophilic and cysteine residues in proteins [ 40 , 41 , 42 ]. Since we have found the “artificial” VL domain in several specific recombinant Abs, its presence does not appear to be critical for the antigen-binding activity of these scFv fragments.…”

Generation of TIM3 inhibitory single‑domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells

“…15596018; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA at 37°C for 15 min using a PrimeScript™ kit (Takara Bio, Inc.). PCR amplification of the VH domains was performed as previously described ( 18 ). The VH fragments were digested with the restriction enzyme Sfi I and cloned into the phage display vector pComb3× (cat.…”

“…Numerous sdAbs are being actively evaluated in clinical trials, and one sdAb drug, caplacizumab (a humanized camelid V HH ), has been recently approved for the treatment of acquired thrombotic thrombocytopenic purpura and thrombosis ( 16 , 17 ). Rabbits have been identified as a convenient model to generate VH domain antibodies via immunization and phage display ( 18 ). In the present study, a similar strategy was adopted to generate TIM3 inhibitory sdAbs that could be combined with chimeric antigen receptor (CAR) T cell therapy to boost CAR T cell efficacy in cancer treatment.…”