Application of Assisted Design of Antibody and Protein Therapeutics (ADAPT) improves efficacy of a Clostridium difficile toxin A single-domain antibody.


Abstract

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.

Submission Details

ID: F8rJ7Mk43

Submitter: Shu-Ching Ou

Submission Date: March 21, 2019, 12:24 p.m.

Version: 1

Publication Details
Sulea T;Hussack G;Ryan S;Tanha J;Purisima EO,Sci Rep (2018) Application of Assisted Design of Antibody and Protein Therapeutics (ADAPT) improves efficacy of a Clostridium difficile toxin A single-domain antibody. PMID:29396522
Additional Information

In silico affinity calculations use fragment (refer to Supplementary Figure S1) instead of full length.

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

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)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.3 A camelid sdAb A26.8 P16154 TOXA_CLODI