Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VHH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (KD) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC50 of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.
Submitter: Shu-Ching Ou
Submission Date: March 21, 2019, 12:24 p.m.
In silico affinity calculations use fragment (refer to Supplementary Figure S1) instead of full length.
|Number of data points||359|
|Proteins||camelid sdAb A26.8|
|Assays/Quantities/Protocols||Experimental Assay: koff ; Experimental Assay: kon ; Experimental Assay: Kd ; Experimental Assay: Tm ; Derived Quantity: SD of Tm ; Derived Quantity: ΔΔG ; Derived Quantity: Kd_WT/Kd_Mutant ; Derived Quantity: SD of Kd ; Derived Quantity: SD of koff ; Derived Quantity: SD of kon ; Computational Protocol: Z-Score|
|Libraries||Variants for sdAb A26.8 ; Variants for Full_TcdA_sdAb ; Variants for Fragment_TcdA_sdAb|
|Structure ID||Release Date||Resolution||Structure Title|
|4DMV||2012-07-18||1.5||Crystal structure of the GT domain of Clostridium difficile Toxin A|
|3HO6||2009-06-23||1.6||Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in clostridium difficile toxin A|
|5UQK||2017-05-10||1.85||Clostridium difficile toxin A (TcdA) glucosyltransferase domain in complex with U2F|
|2F6E||2005-12-20||1.85||Clostridium difficile Toxin A C-terminal fragment 1 (TcdA-f1)|
|5UQL||2017-05-10||1.97||Clostridium difficile toxin A (TcdA) glucosyltransferase domain in complex with U2F|
|2G7C||2006-04-18||2.0||Clostridium difficile Toxin A Fragment Bound to aGal(1,3)bGal(1,4)bGlcNAc|
|2QJ6||2008-07-15||2.5||Crystal structure analysis of a 14 repeat C-terminal fragment of toxin TcdA in Clostridium difficile|
|4DMW||2012-07-18||2.5||Crystal structure of the GT domain of Clostridium difficile toxin A (TcdA) in complex with UDP and Manganese|
|5UMI||2017-07-19||3.23||Clostridium difficile TcdA-CROPs bound to PA50 Fab|
|4R04||2016-01-06||3.26||Clostridium difficile Toxin A (TcdA)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|96.3||A||camelid sdAb A26.8||P16154||TOXA_CLODI|