Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Abstract

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.

Properties and pervaporation performance of poly(vinyl alcohol) membranes crosslinked with various dianhydrides

“…A static pervaporation cell of laboratory scale was used for the pervaporation test, and its schematic design was depicted elsewhere . The membrane was fixed in a stainless steel permeation cell with an effective area of 13.5 cm 2 .…”

The Origin of CDR H3 Structural Diversity

“…Library screening and directed evolution techniques have enabled the successful production of engineered antibodies used for sensors and assays as well as novel therapeutics (Buss et al, 2012; Lequin, 2005; Lu et al, 2002; Xu et al, 2013). However, discovery and development of such antibodies remains challenging.…”

Blind prediction performance of RosettaAntibody 3.0: Grafting, relaxation, kinematic loop modeling, and full CDR optimization

“…Templated regions were predicted to about 1 Å RMSD, and CDR H3 loops were predicted to about 3 Å RMSD. Since that time, efforts in the antibody structure prediction field have included updated databases, additional examination of the V L –V H orientation, reassessment of canonical loop clusters, designs of antibodies for thermal resistance, and noncanonical residue antigen crosslinking . In addition, progress has been made in ab initio loop modeling .…”

“…Since that time, efforts in the antibody structure prediction field have included updated databases, 42 additional examination of the V L -V H orientation, 43 reassessment of canonical loop clusters, 26 designs of antibodies for thermal resistance, 8 and noncanonical residue antigen crosslinking. 44 In addition, progress has been made in ab initio loop modeling. [45][46][47][48] In response to these developments, a second antibody modeling assessment was organized this year.…”